No / Although Streptococcus agalactiae, the group B Streptococcus, is a leading cause of invasive neonatal disease worldwide the molecular basis of its virulence is still poorly understood. To investigate the role of lipoproteins in the physiology and interaction of this pathogen with host cells, we generated a mutant S. agalactiae strain (A909DeltaLgt) deficient in the Lgt enzyme and thus unable to lipidate lipoprotein precursors (pro-lipoproteins). The loss of pro-lipoprotein lipidation did not affect the viability of S. agalactiae or its growth in several different media, including cation-depleted media. The processing of two well-characterized lipoproteins, but not a non-lipoprotein, was clearly shown to be aberrant in A909DeltaLgt. The mutant strain was shown to be more sensitive to oxidative stress in vitro although the molecular basis of this increased sensitivity was not apparent. The inactivation of Lgt also resulted in changes to the bacterial cell envelope, as demonstrated by reduced retention of both the group B carbohydrate and the polysaccharide capsule and a statistically significant reduction (P=0.0079) in A909DeltaLgt adherence to human endothelial cells of fetal origin. These data confirm that failure to process lipoproteins correctly has pleiotropic effects that may be of significance to S. agalactiae colonization and pathogenesis.

Yes / Background: Group B Streptococcus (GBS) is a leading cause of morbidity and mortality in infants in both developed and developing countries. To our knowledge, only a few studies have been reported the clinical features, treatment and outcomes of the GBS disease in China. The severity of neonatal GBS disease in China remains unclear. Population-based surveillance in China is therefore required. Methods: We retrospectively collected data of <3 months old infants with culture-positive GBS in sterile samples from three large urban tertiary hospitals in South China from Jan 2011 to Dec 2014. The GBS isolates and their antibiotic susceptibility were routinely identified in clinical laboratories in participating hospitals. Serotyping and multi-locus sequence typing (MLST) were also conducted for further analysis of the neonatal GBS disease. Results: Total 70 cases of culture-confirmed invasive GBS infection were identified from 127,206 live births born in studying hospitals, giving an overall incidence of 0.55 per 1000 live births (95% confidence interval [CI] 0.44–0.69). They consisted of 49 with early-onset disease (EOD, 0.39 per 1000 live births (95% CI 0.29–0.51)) and 21 with late-onset disease (LOD, 0.17 per 1000 live births (95% CI 0.11–0.25)). The incidence of EOD increased significantly over the studying period. Five infants (4 EOD and 1 LOD) died before discharge giving a mortality rate of 7.1% and five infants (7.1%, 2 EOD and 3 LOD) had neurological sequelae. Within 68 GBS isolates from GBS cases who born in the studying hospitals or elsewhere, serotype III accounted for 77.9%, followed by Ib (14.7%), V (4.4%), and Ia (2.9%). MLST analysis revealed the presence of 13 different sequence types among the 68 GBS isolates and ST-17 was the most frequent sequence type (63.2%). All isolates were susceptible to penicillin, ceftriaxone, vancomycin and linezolid, while 57.4% and 51.5% were resistant to erythromycin and clindamycin, respectively. Conclusions: This study gains the insight into the spectrum of GBS infection in south China which will facilitate the development of the guidance for reasonable antibiotics usage and will provide evidence for the implementation of potential GBS vaccines in the future. / Supported by medical and health science and technology projects of Health and Family Planning Commission of Guangzhou Municipality (grant number 20151A010034) and Guangdong provincial science and technology planning projects (grant number 2014A020212520).

Le Streptocoque de groupe B (GBS) est un important agent d’infection invasive pouvant mener à la mort et demeure la cause principale de septicémie néonatale à ce jour. Neuf sérotypes ont été officiellement décrits basés sur la composition de la capsule polysaccharidique (CPS). Parmi ces sérotypes, le type III est considéré le plus virulent et fréquemment associé aux maladies invasives graves, telle que la méningite. Malgré que plusieurs recherches aient été effectuées au niveau des interactions entre GBS type III et les cellules du système immunitaire innées, aucune information n’est disponible sur la régulation de la réponse immunitaire adaptative dirigée contre ce dernier. Notamment, le rôle de cellules T CD4+ dans l’immuno-pathogenèse de l’infection causée par GBS n’a jamais été étudié. Dans cet étude, trois différents modèles murins d’infection ont été développé pour évaluer l’activation et la modulation des cellules T CD4+ répondantes au GBS de type III : ex vivo, in vivo, et in vitro. Les résultats d’infections ex vivo démontrent que les splénocytes totaux répondent à l’infection en produisant des cytokines de type-1 pro-inflammatoires. Une forte production d’IL-10 accompagne cette cascade inflammatoire, probablement dans l’effort de l’hôte de maintenir l’homéostasie. Les résultats démontrent aussi que les cellules T sont activement recrutées par les cellules répondantes du système inné en produisant des facteurs chimiotactiques, tels que CXCL9, CXCL10, et CCL3. Plus spécifiquement, les résultats obtenus à partir des cellules isolées T CD4+ provenant des infections ex vivo ou in vivo démontrent que ces cellules participent à la production d’IFN-γ et de TNF-α ainsi que d’IL-2, suggérant un profil d’activation Th1. Les cellules isolées T CD4+ n’étaient pas des contributeurs majeurs d’IL-10. Ceci indique que cette cytokine immuno-régulatrice est principalement produite par les cellules de l’immunité innée de la rate de souris infectées. Le profil Th1 des cellules T CD4+ a été confirmé en utilisant un modèle in vitro. Nos résultats démontrent aussi que la CPS de GBS a une role immuno-modulateur dans le développement de la réponse Th1. En résumé, cette étude adresse pour la première fois, la contribution des cellules T CD4+ dans la production d’IFN-γ lors d’une infection à GBS et donc, dans le développement d’une réponse de type Th1. Ces résultats renforcent d’avantage le rôle central de cette cytokine pour un control efficace des infections causées par ce pathogène. / Group B Streptococcus (GBS) is an important agent of life-threatening invasive infections and remains the leading cause of neonatal sepsis to this day. Nine serotypes have been officially described based on capsular polysaccharide (CPS) composition. Among them, capsular type III is considered one of the most virulent and frequently associated with severe invasive diseases, such as meningitis. Although extensive research has been done on the interactions between GBS type III and various cells of the innate immune system, no information is available on the regulation of the adaptive immune response against this pathogen. In particular, the role of CD4+ T cells in the immuno-pathogenesis of the infection caused by GBS has never been assessed. In this study, three different models of murine infection were developed to evaluate activation and modulation of responding CD4+ T cells against GBS type III: ex vivo, in vivo, and in vitro. Ex vivo analysis of total splenocytes showed that GBS induces the release of type-1 pro-inflammatory cytokines. A strong IL-10 production follows this inflammatory cascade, indicating the host effort to maintain homeostasis. Results also indicate that T cells were actively recruited by responding innate immune cells via the release of chemotactic factors such as CXCL9, CXCL10, and CCL3. More specifically, results obtained from isolated CD4+ T cells from ex vivo or in vivo infections showed that they actively participate in the production of IFN-γ and TNF-α, as well as IL-2, suggesting a Th1 profile of activation. On the other hand, isolated CD4+ T cells were not main sources of IL-10. This observation suggests that this immuno-regulatory cytokine is produced mainly by cells of the spleen innate immune system of infected animals. The CD4+ Th1 cell profile was confirmed using an in vitro model of infection. Our results also suggest that the GBS CPS plays an immuno-modulatory role in the development of a Th1 response. In summary, this study addresses for this first time the contribution of CD4+ T cells in IFN-γ production during GBS infection, and thus, in the development of a Th1 response. Our data further highlight the central role of this cytokine for effective control of GBS infections.

Le Streptocoque de groupe B (GBS) est un important agent d’infection invasive pouvant mener à la mort et demeure la cause principale de septicémie néonatale à ce jour. Neuf sérotypes ont été officiellement décrits basés sur la composition de la capsule polysaccharidique (CPS). Parmi ces sérotypes, le type III est considéré le plus virulent et fréquemment associé aux maladies invasives graves, telle que la méningite. Malgré que plusieurs recherches aient été effectuées au niveau des interactions entre GBS type III et les cellules du système immunitaire innées, aucune information n’est disponible sur la régulation de la réponse immunitaire adaptative dirigée contre ce dernier. Notamment, le rôle de cellules T CD4+ dans l’immuno-pathogenèse de l’infection causée par GBS n’a jamais été étudié. Dans cet étude, trois différents modèles murins d’infection ont été développé pour évaluer l’activation et la modulation des cellules T CD4+ répondantes au GBS de type III : ex vivo, in vivo, et in vitro. Les résultats d’infections ex vivo démontrent que les splénocytes totaux répondent à l’infection en produisant des cytokines de type-1 pro-inflammatoires. Une forte production d’IL-10 accompagne cette cascade inflammatoire, probablement dans l’effort de l’hôte de maintenir l’homéostasie. Les résultats démontrent aussi que les cellules T sont activement recrutées par les cellules répondantes du système inné en produisant des facteurs chimiotactiques, tels que CXCL9, CXCL10, et CCL3. Plus spécifiquement, les résultats obtenus à partir des cellules isolées T CD4+ provenant des infections ex vivo ou in vivo démontrent que ces cellules participent à la production d’IFN-γ et de TNF-α ainsi que d’IL-2, suggérant un profil d’activation Th1. Les cellules isolées T CD4+ n’étaient pas des contributeurs majeurs d’IL-10. Ceci indique que cette cytokine immuno-régulatrice est principalement produite par les cellules de l’immunité innée de la rate de souris infectées. Le profil Th1 des cellules T CD4+ a été confirmé en utilisant un modèle in vitro. Nos résultats démontrent aussi que la CPS de GBS a une role immuno-modulateur dans le développement de la réponse Th1. En résumé, cette étude adresse pour la première fois, la contribution des cellules T CD4+ dans la production d’IFN-γ lors d’une infection à GBS et donc, dans le développement d’une réponse de type Th1. Ces résultats renforcent d’avantage le rôle central de cette cytokine pour un control efficace des infections causées par ce pathogène. / Group B Streptococcus (GBS) is an important agent of life-threatening invasive infections and remains the leading cause of neonatal sepsis to this day. Nine serotypes have been officially described based on capsular polysaccharide (CPS) composition. Among them, capsular type III is considered one of the most virulent and frequently associated with severe invasive diseases, such as meningitis. Although extensive research has been done on the interactions between GBS type III and various cells of the innate immune system, no information is available on the regulation of the adaptive immune response against this pathogen. In particular, the role of CD4+ T cells in the immuno-pathogenesis of the infection caused by GBS has never been assessed. In this study, three different models of murine infection were developed to evaluate activation and modulation of responding CD4+ T cells against GBS type III: ex vivo, in vivo, and in vitro. Ex vivo analysis of total splenocytes showed that GBS induces the release of type-1 pro-inflammatory cytokines. A strong IL-10 production follows this inflammatory cascade, indicating the host effort to maintain homeostasis. Results also indicate that T cells were actively recruited by responding innate immune cells via the release of chemotactic factors such as CXCL9, CXCL10, and CCL3. More specifically, results obtained from isolated CD4+ T cells from ex vivo or in vivo infections showed that they actively participate in the production of IFN-γ and TNF-α, as well as IL-2, suggesting a Th1 profile of activation. On the other hand, isolated CD4+ T cells were not main sources of IL-10. This observation suggests that this immuno-regulatory cytokine is produced mainly by cells of the spleen innate immune system of infected animals. The CD4+ Th1 cell profile was confirmed using an in vitro model of infection. Our results also suggest that the GBS CPS plays an immuno-modulatory role in the development of a Th1 response. In summary, this study addresses for this first time the contribution of CD4+ T cells in IFN-γ production during GBS infection, and thus, in the development of a Th1 response. Our data further highlight the central role of this cytokine for effective control of GBS infections.

Tyrimo tikslas – nustatyti nėščių moterų B grupės β hemolizinio streptokoko (BGS) ir Escherichia coli (E.coli) nešiojimo bei naujagimių kolonizacijos dažnumą ir įvertinti šių mikroorganizmų įtaką naujagimių ankstyvai infekcijai atsirasti. Metodika. Perspektyvinis momentinis stebėjimo tyrimas vykdytas Kauno medicinos universiteto Akušerijos ir ginekologijos bei Neonatologijos klinikose. Moterims du atskiri pasėliai iš makšties apatinio trečdalio ir išangės buvo paimti 35-37 nėštumo savaitę arba gimdymo metu, o naujagimiams - iš ausies išorinės landos bei nosiaryklės per 5 – 15 min. po gimimo. Išskirtų BGS serotipavimas atliktas naudojant 9 specifinius antiserumus, o jautrumas antibiotikams nustatytas diskų difuzijos metodu pagal klinikinių laboratorijų standartus nustatančio komiteto (NCCLS) rekomendacijas. Rezultatai. Šimtas keturiasdešimt aštuonioms moterims iš 970 (15,3 proc.) buvo nustatytas BGS nešiojimas, o 193 moterims (19,9 proc.) - E.coli nešiojimas. Naujagimių BGS ir E.coli kolonizacijos dažnumas buvo 6,4 proc. ir 14,4 proc., o vertikalaus pernešimo – atitinkamai 28,4 ir 24,4 procentai. Moterims ir naujagimiams dažniausiai identifikuotas III ir Ia serotipo BGS. Bendras naujagimių įgimtos infekcijos dažnumas buvo 37,5 atvejai iš 1000 naujagimių, o BGS sukeltos infekcijos dažnumas - 3,6 atvejai iš 1000 naujagimių. Nebuvo nė vieno E.coli sukeltos ankstyvos naujagimių infekcijos atvejo. Klinikinis sepsis diagnozuotas 5 kartus dažniau nei mikrobiologiniais tyrimais... [toliau žr. visą tekstą] / Objective - to examine the prevalence of maternal and neonatal colonization of group B streptococcus (GBS) and Escherichia coli (E.coli) in our area, and to evaluate the colonization impact on early onset neonatal infections as a whole. Methods. A prospective cross-sectional study carried out at the Department of Obstetrics and Gynaecology and the Department of Neonatology of Kaunas University Hospital. Samples were collected from the lower vagina and the anorectum of pregnant women at 35-37 weeks of gestation or at delivery and the ear canal as well as throat of the neonates within 5 – 15 min of their lives. The distribution of serotypes of the GBS identified was determined using specific antisera and antimicrobial susceptibility was investigated by disc-diffusion method as described by National Committee for Clinical Laboratory Standards (NCCLS). Results. GBS carriage was detected in 148 (15.3%) of 970 women screened whereas E.coli colonisation was found in 193 (19.9%) of the women studied. The overall GBS and E.coli neonatal colonization rates were 6.4% and 14.4%; vertical transmission rates - 28.4% and 24.4%, respectively. The most common GBS serotypes were III and Ia. The overall incidence of early onset neonatal infection was estimated to be 37.5 per 1000 live birth and the incidence of early onset GBS disease in newborns – 3.6 per 1000 live birth. There was no case of early neonatal E. coli infection. The incidence of clinical sepsis in neonates was 5 fold higher that... [to full text]

Résumé : Le streptocoque de groupe B (SGB) est une bactérie commensale présente dans le tractus génito-urinaire de 10 à 30 % des femmes enceintes en santé. Ce pathogène est responsable de chorioamnionite, associée aux naissances prématurées et aux dommages cérébraux du nouveau-né. Les infections durant la grossesse, la chorioamnionite et la prématurité sont associées au développement de troubles du spectre de l’autisme. Notre hypothèse est qu’une exposition subclinique au SGB induit une réponse inflammatoire maternofoetale, menant à des troubles neurodéveloppementaux et comportementaux de type autistique dans la progéniture. L’objectif principal est d’étudier, à l’aide d’un nouveau modèle animal (rat) préclinique, les impacts d’une exposition au SGB en période prénatale sur le développement cérébral de la progéniture. Les rates Lewis gestantes sont injectées au jour de gestation 19 avec une dose de SGB de sérotype Ia (108 UFC/100µl) ou de saline. La réponse inflammatoire placentaire est caractérisée par immunohistochimie. Des tests comportementaux sont effectués entre les jours postnataux 7 et 40 afin d’évaluer la communication, le comportement exploratoire, l’intégration sensorielle et les interactions sociales. Une chorioamnionite dichotomique selon le genre est observée dans les placentas exposés au SGB, via une infiltration de cellules polymorphonucléaires. Cette infiltration est significativement plus proéminente dans les placentas associés aux fœtus mâles que ceux des fœtus femelles. Les mâles exposés au SGB ont un amincissement de la substance blanche cérébrale adjacente aux ventricules latéraux élargis. La progéniture mâle exposée au SGB présente des anomalies comportementales associées aux traits cardinaux des troubles du spectre de l’autisme, soit des déficits au niveau de la communication, des interactions sociales, du traitement de l’information sensorielle ainsi qu’au niveau d’autres comorbidités classiques de l’autisme, comme l’hyperactivité. Ces données démontrent pour la première fois que l’activation immune maternelle induite par l’infection au SGB joue un rôle dans l’induction d’anomalies neurodéveloppementales récapitulant celles observées chez les patients autistes, incluant la dichotomie de genre et le phénotype neurocomportemental. Ces résultats fournissent de nouvelles évidences en faveur du rôle dans la physiopathologie de l’autisme d’un facteur environnemental commun, et modifiable, d’inflammation gestationnelle. / Abstract : Group B Streptococcus (GBS) is a commensal bacterium present in the vagina of 10 to 30% of healthy pregnant women. GBS is responsible for chorioamnionitis, which can cause preterm birth and cerebral injuries in the newborn most often in the absence of maternofetal pathogen translocation. Maternal infection, chorioamnionitis and preterm birth are associated to autism spectrum disorders (ASD) in the progeny. Our hypothesis is that GBS-induced gestational infection induces a maternofetal inflammatory response leading to neurodevelopmental impairments and ASD-like behaviour in the offspring. Our goal was to study, with a new preclinical animal model, the impacts of GBS-induced gestational inflammation on the neurodevelopmental features in the offspring. We characterized GBS-induced placental and neurobehavioural outcomes. Dams were exposed at gestational day 19 to live GBS or saline. The placental inflammatory response was studied by immunohistochemistry. Behavioural tests were performed between postnatal days 7 and 40 to assess communication, exploratory abilities, sensory integration and social interactions. GBS-exposed placentas displayed chorioamnionitis featured by infiltration of polymorphonuclear cells, which was significantly more prominent in males than in females. GBS-exposed males showed a reduced thickness of periventricular white matter. Male offspring exposed to GBS had early onset of cardinal ASD-like traits affecting social interaction, communication (ultrasonic vocalizations), treatment of sensory information (prepulse inhibition), preference toward mother cue (nest-seeking), and some other classic ASD comorbidities such as hyperactivity (open field). Overall, these data show for the first time that maternal immune activation due to live GBS plays a key role in the induction of neurodevelopmental abnormalities recapitulating those of human ASD, including gender dichotomy and neurobehavioural phenotype. These results provide new evidence in favor of the role of a common and modifiable infectious/inflammatory environmental factor in human ASD pathophysiology.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Streptococcus agalactiae, ou Streptococcus do grupo B (GBS), é um importante patógeno oportunista que causa pneumonia, sepse e meningite em recém-nascidos e infecções em adultos imunocomprometidos. O pulmão aparentemente é o portal de entrada para o EGB na corrente sanguínea o que pode evoluir para uma septicemia. Os mecanismos de virulência relevantes envolve a habilidade do EGB em penetrar e sobreviver intracelularmente em células hospedeiras. Neste trabalho, foram analisados os mecanismos moleculares da apoptose epitelial induzida pelo EGB, e a produção de óxido nítrico (NO) e espécies reativas de oxigênio (ROS) em células epiteliais respiratórias A549 durante a infecção por EGB. Todas as amostras de EGB exibiram a capacidade de aderir e invadir células A549. A sobrevivência intracelular do EGB em células A549 ocorreu durante 24 h de incubação sem replicação do patógeno. No entanto, a amsotra 88641-V isolada de vagina não sobreviveu após 0,5 h de interação. O EGB promoveu a perda de viabilidade do epitélio durante a infecção. As alterações morfológicas em células A549 infectadas com o EGB incluem arredondamento celular, condensação nuclear, encolhimento celular e perda de contato célula-célula e célula-substrato. A dupla marcação AV/IP revelou que amostras de EGB sorotipo III induziram apoptose enquanto amostras do sorotipo V induziram morte celular semelhante a necrose em células A549. Caspase-3 foi ativada durante a apoptose induzida por EGB em células epiteliais. No entanto, a ativação de caspases-8 e -9 foi detectada apenas para a amostra 88641-V e as amostras EGB do sorotipo III, respectivamente. Experimentos comparativos de Immunoblotting revelaram que o EGB induziu um aumento da expressão Bim, uma proteína pró-apoptótica e diminuiu a expressão de Bcl-2 e Bcl-xL, proteínas anti-apoptóticas. As células A549 apresentaram perda de potencial de membrana mitocondrial &#916;&#968;m e co-localização com o Bax. Ensaio de espectrometria de massa identificou a proteína PI-2a, uma proteína estrutural de pili, que exibe atividade carboxipepdidase. Descobrimos que os dois sorotipos (III e V) induziram a produção ROS e NO em células A549. Em conclusão, a apoptose induzida pelo EGB em células A549 é um mecanismo importante de virulência, resultando na destruição de tecidos, escape do sistema imune do hospedeiro com espalhamento bacteriano e, em consequência, a doença invasiva ou uma infecção sistémica. / Streptococcus agalactiae, or group B Streptococcus (GBS), is an important opportunistic pathogen that causes pneumonia, sepsis, and meningitis in neonates and severe diseases in immunocompromised adults. The lung is the apparent portal of entry for GBS into the bloodstream, after which septicemia may ensue. A relevant virulence mechanism involves the ability of GBS to penetrate and to survive intracellularly within these host cells. In this work, we analyzed the molecular mechanisms of GBS-induced epithelial apoptosis, and nitric oxide (NO) and reactive oxygen species (ROS) production by lung epithelial cell line A549 cells during infection with GBS. All GBS exhibited the ability to adhere and to invade A549 cells. The survival of GBS within A549 cells without replication was shown during 24 h incubation. However, the 88641-V strain isolated from vagina did not survive after 0.5 h of interaction. GBS promoted the loss of viability of the epithelium during infection. The morphological changes in A549 cells infected with GBS included cell rounding, nuclear condensation, cellular shrinkage and loss of cell-cell contact and cell-substrate. The double staining AV / IP revealed that GBS serotype III induced apoptosis while GBS serotype V induced like necrosis cell death in A549 cells. Caspase-3 was activated during GBS-induced endothelial apoptosis. However, activation of caspases-8 and -9 was detected only by GBS 88641-V and GBS-III, respectively. Comparative immunoblotting experiments revealed that GBS induced an increasing pro-apoptotic Bim expression and decreasing anti-apoptotic Bcl-2 and Bcl-XL expression. A549 cells exhibited loss of mitochondrial membrane potential &#916;&#968;m with Bax colocalization. Mass spectrometry assay identified protein PI-2a, a structural protein pili, which exhibit carboxipepdidase activity. We found that both serotypes (III and V) induced ROS and NO production in A549 cells. In conclusion, apoptosis of A549 cell induced by GBS is an important virulence mechanism resulting in tissue destruction, escape from the host immune system with bacterial spreading and, in consequence, invasive disease or systemic infection.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Streptococcus agalactiae, ou Streptococcus do grupo B (GBS), é um importante patógeno oportunista que causa pneumonia, sepse e meningite em recém-nascidos e infecções em adultos imunocomprometidos. O pulmão aparentemente é o portal de entrada para o EGB na corrente sanguínea o que pode evoluir para uma septicemia. Os mecanismos de virulência relevantes envolve a habilidade do EGB em penetrar e sobreviver intracelularmente em células hospedeiras. Neste trabalho, foram analisados os mecanismos moleculares da apoptose epitelial induzida pelo EGB, e a produção de óxido nítrico (NO) e espécies reativas de oxigênio (ROS) em células epiteliais respiratórias A549 durante a infecção por EGB. Todas as amostras de EGB exibiram a capacidade de aderir e invadir células A549. A sobrevivência intracelular do EGB em células A549 ocorreu durante 24 h de incubação sem replicação do patógeno. No entanto, a amsotra 88641-V isolada de vagina não sobreviveu após 0,5 h de interação. O EGB promoveu a perda de viabilidade do epitélio durante a infecção. As alterações morfológicas em células A549 infectadas com o EGB incluem arredondamento celular, condensação nuclear, encolhimento celular e perda de contato célula-célula e célula-substrato. A dupla marcação AV/IP revelou que amostras de EGB sorotipo III induziram apoptose enquanto amostras do sorotipo V induziram morte celular semelhante a necrose em células A549. Caspase-3 foi ativada durante a apoptose induzida por EGB em células epiteliais. No entanto, a ativação de caspases-8 e -9 foi detectada apenas para a amostra 88641-V e as amostras EGB do sorotipo III, respectivamente. Experimentos comparativos de Immunoblotting revelaram que o EGB induziu um aumento da expressão Bim, uma proteína pró-apoptótica e diminuiu a expressão de Bcl-2 e Bcl-xL, proteínas anti-apoptóticas. As células A549 apresentaram perda de potencial de membrana mitocondrial &#916;&#968;m e co-localização com o Bax. Ensaio de espectrometria de massa identificou a proteína PI-2a, uma proteína estrutural de pili, que exibe atividade carboxipepdidase. Descobrimos que os dois sorotipos (III e V) induziram a produção ROS e NO em células A549. Em conclusão, a apoptose induzida pelo EGB em células A549 é um mecanismo importante de virulência, resultando na destruição de tecidos, escape do sistema imune do hospedeiro com espalhamento bacteriano e, em consequência, a doença invasiva ou uma infecção sistémica. / Streptococcus agalactiae, or group B Streptococcus (GBS), is an important opportunistic pathogen that causes pneumonia, sepsis, and meningitis in neonates and severe diseases in immunocompromised adults. The lung is the apparent portal of entry for GBS into the bloodstream, after which septicemia may ensue. A relevant virulence mechanism involves the ability of GBS to penetrate and to survive intracellularly within these host cells. In this work, we analyzed the molecular mechanisms of GBS-induced epithelial apoptosis, and nitric oxide (NO) and reactive oxygen species (ROS) production by lung epithelial cell line A549 cells during infection with GBS. All GBS exhibited the ability to adhere and to invade A549 cells. The survival of GBS within A549 cells without replication was shown during 24 h incubation. However, the 88641-V strain isolated from vagina did not survive after 0.5 h of interaction. GBS promoted the loss of viability of the epithelium during infection. The morphological changes in A549 cells infected with GBS included cell rounding, nuclear condensation, cellular shrinkage and loss of cell-cell contact and cell-substrate. The double staining AV / IP revealed that GBS serotype III induced apoptosis while GBS serotype V induced like necrosis cell death in A549 cells. Caspase-3 was activated during GBS-induced endothelial apoptosis. However, activation of caspases-8 and -9 was detected only by GBS 88641-V and GBS-III, respectively. Comparative immunoblotting experiments revealed that GBS induced an increasing pro-apoptotic Bim expression and decreasing anti-apoptotic Bcl-2 and Bcl-XL expression. A549 cells exhibited loss of mitochondrial membrane potential &#916;&#968;m with Bax colocalization. Mass spectrometry assay identified protein PI-2a, a structural protein pili, which exhibit carboxipepdidase activity. We found that both serotypes (III and V) induced ROS and NO production in A549 cells. In conclusion, apoptosis of A549 cell induced by GBS is an important virulence mechanism resulting in tissue destruction, escape from the host immune system with bacterial spreading and, in consequence, invasive disease or systemic infection.

Orientador: Renato Passini Junior / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-04T01:25:35Z (GMT). No. of bitstreams: 1 Nomura_MarceloLuis_D.pdf: 838174 bytes, checksum: a795b39457b62a18ecb17c4609ece12d (MD5) Previous issue date: 2004 / Resumo: Objetivos: Identificar a taxa de prevalência e fatores de risco de colonização materna por estreptococo do grupo B (EGB) em gestantes com trabalho de parto prematuro (TPP) e/ou ruptura prematura pré-termo de membranas (RPM). Métodos: Foram colhidos dois swabs anais e vaginais de 203 gestantes atendidas no CAISM-UNICAMP. Um swab de cada local foi colocado em meio de transporte e enviados para cultura em placas de ágar-sangue, os outros dois foram incubados por 24 horas em meio de Todd-Hewitt para posterior semeadura em placas de ágar-sangue. Resultados: A prevalência de colonização materna por EGB foi de 27,6% (56 gestantes). As taxas de colonização por diagnóstico foram 34,7% para RPM, 25,2% para TPP e 17,8% para TPP + RPM. As variáveis raça branca, baixo nível de escolaridade e infecção urinária foram associadas a maiores taxas de colonização na análise multivariada. A presença de infecção urinária foi a única variável significativamente associada à colonização materna na análise multivariada. A taxa de detecção do estreptococo do grupo B foi significativamente maior com o uso do meio seletivo e com a associação de coleta de culturas anais e vaginais. A taxa de colonização neonatal foi de 3,1%. Ocorreram dois casos de sepse precoce por EGB nesta amostra, com prevalência estimada de 10,8 casos por mil nascidos vivos e mortalidade de 50%. Conclusão: A amostra avaliada apresenta altas taxas de colonização materna por Streptococcus agalactiae. É necessário o uso de meio de cultura seletivo e a associação de culturas anorretais e vaginais para aumentar a taxa de detecção do EGB. A incidência de sepse neonatal precoce foi elevada nesta população / Abstract: Objective: to study group B streptococcus maternal colonization rates and risk factors in women with preterm labor (PTL) and preterm premature rupture of membranes (PROM). Methods: Vaginal and anal swabs (two of each) were colected from 203 women followed at CAISM-UNICAMP. One of each swab was placed in transport media and then cultured in blood-agar plates, the other swabs were incubated in Todd-Hewitt selective media for 24 hours and then subcultured in blood-agar plates. Results: Maternal colonization rate was 27.6% (56 women). Colonization rates by admission diagnosis were 34.7% in PROM, 25.2% in PTL and 17.8% in PTL and PROM. White race, less than elementary education level and urinary tract infection were associated with maternal colonization in the univariate analysis. Urinary tract infection was the only variable associated with maternal colonization in a multivariate analysis. GBS detection rates were significantly higher with the use of selective culture media and with sampling of both vaginal and anorectal sites. Neonatal colonization rate was 3.1%. There were two cases of early-onset neoanatal sepsis caused by GBS, with an estimated prevalence of 10.8 cases per thousand live borns and a mortality rate of 50%. Conclusions: This sample of women had high GBS colonization rates. The use of selective culture media and collection of both anal and vaginal samples is necessary in order to maximize GBS detection rates. Early-onset neonatal sepsis incidence was high in this population / Doutorado / Tocoginecologia / Doutor em Tocoginecologia

BACKGROUND AND OBJECTIVES: Streptococcus agalactiae (Group B Streptococcus, GBS) is the leading cause of neonatal infections and deaths in human. It can also cause infections in pregnant women and non-pregnant adults. Penicillin and ampicillin are antibiotics of choice for the treatment of GBS infections. Erythromycin and clindamycin are used as alternative therapy in penicillin allergic patients, however resistance to these agents has been increasingly observed. This present study was undertaken to determine the colonization rate of GBS, susceptibility profile and the mechanism of antibiotic resistance in pregnant women and their babies at Dr. George Mukhari Academic Hospital in Pretoria. METHODS: Rectal and vaginal swabs were collected from pregnant women; ear and umbilical swabs from newborns over an 11 month period. Samples were cultured on selective media (CNA agar and Todd-Hewitt broth) and GBS positively identified using morphological and biochemical tests including Gram staining, hemolytic activity, catalase test, bile esculin, CAMP test and Latex agglutination test. The susceptibility testing was done using the Kirby-Bauer and E-test methods. The D-test method was used to determine the inducible clindamycin resistance. Multiplex PCR with were used to detect different genes coding for resistance. RESULTS: Out of the 413 patients evaluated, 128 (30.9%) were positive with GBS. All isolates were sensitive to penicillin and ampicillin. Erythromycin and clindamycin resistance was 21.1% and 17.2% respectively; of which 69% harbouring constitutive MLBB, 17.4% inducible MLSB. The alteration of ribosomal target encoded by ermB genes was the commonest mechanism of resistance observed in 55% of isolates, 38% of isolates had both ermB and linB genes and efflux pump mediated by mefA genes was detected in one of isolates. Conclusion: This study reaffirms the appropriateness of penicillin as the antibiotic of choice for treating GBS infection. However it raises the challenges of resistance to the macrolides and lincosamides. More GBS treatment options for penicillin allergic patients need to be researched. / Health Studies / M.Sc. (Life Sciences (Microbiology))