Contribuição para a caracterização da população de Streptococcus pneumoniae causadora de infecções graves

Tese de doutoramento em Ciências da Saúde (Microbiologia), apresentada à Universidade de Lisboa através da Faculdade de Medicina, 2008A espécie Streptococcus pneumoniae (pneumococo) é um agente potencialmente patogénico que pode viver na nasofaringe assintomaticamente ou causar infecções invasivas e não invasivas no Homem. Está associado a uma elevada taxa de morbilidade e mortalidade, sobretudo em crianças com menos de 2 anos. Face à importância desta bactéria para a saúde humana foram estudados dois temas. O primeiro é a variação de fase, um mecanismo de regulação de factores de virulência usado por S. pneumoniae, cuja importância em infecções no Homem está ainda por esclarecer. O segundo, diz respeito à estrutura da população responsável por infecções invasivas antes e depois da disponibilização em Portugal da vacina 7-valente. a) Recentemente foi descrita a variação de fase nos pneumococos. Segundo o modelo proposto o pneumococo pode reverter espontaneamente entre a forma opaca e a transparente. As formas transparentes têm sido associadas a uma colonização mais eficiente em oposição às opacas, enquanto que as formas opacas são as únicas capazes de causar bacteriémia persistente num modelo murino. A maioria destas experiências foram restritas a apenas algumas estirpes com um contexto genético desconhecido e expressando apenas 6 dos 91 tipos capsulares reconhecidos nos pneumococos. Estas observações não esclarecem se a variação de fase é um fenómeno restrito a alguns serótipos ou perfis genéticos ou se é uma propriedade de todos os pneumococos. Se a variação de fase fosse uma propriedade restrita a alguns clones isso poderia explicar o seu sucesso epidemiológico. De modo a resolver esta questão, estudou-se a variação de fase numa colecção diversificada de estirpes causadoras de infecções invasivas previamente caracterizadas quanto ao serótipo e perfil genómico. Foram analisadas 304 estirpes responsáveis por infecções invasivas que expressavam os 10 serótipos mais prevalentes. A visualização do fenótipo foi feita recorrendo a uma lupa diascópica, através da passagem de luz oblíqua num meio transparente. Verificou-se que a maioria (52,0%) das estirpes responsáveis por infecções invasivas eram opacas e apenas 26,0% eram transparentes, o que está de acordo com a variação de fase ter um papel na infecção pneumocócica no Homem. Por outro lado, observou-se uma notória diferença na prevalência de cada fenótipo em cada serótipo. Por isso tentou-se estabelecer uma associação entre o serótipo e a opacidade, estimando o odds ratio empírico, com referência a todos os outros serótipos, específico para cada serótipo e um intervalo de confiança de 95%. Observou-se que os serótipos 1, 4 e 23F estavam mais associados ao fenótipo opaco do que o espectável, enquanto os serótipos 3 e 14 estavam mais associados ao fenótipo transparente. Esta associação pode, em parte, explicar as diferenças epidemiológicas observadas entre os serótipos, como a preferência para colonizarem a nasofaringe ou para serem mais frequentemente isolados de casos de infecção invasiva. De acordo com o esperado, a frequência de transição de fase variou de 10-3 a 10-6, entre o fenótipo opaco, transparente e intermédio. Nas experiências sobre a estabilidade dos fenótipos, garantiu-se a manutenção do fenótipo dominante no crescimento e nas passagens laboratoriais in vitro. Estabeleceu-se a variação de fase num modelo murino de infecção. Neste modelo observou-se que os pneumococos recuperados do sangue dos ratinhos, que morreram de infecção pneumocócica, apresentaram sempre o fenótipo opaco, independentemente do fenótipo previamente administrado. Isto comprovou que, neste modelo, os variantes opacos são mais bem sucedidos a causar bacteriémia do que os transparentes. De modo a relacionar a presença de certas proteínas de superfície com opacidade quantificou-se a actividade da enzima ß-galactosidase e verificou-se que a maioria dos variantes transparentes tinham uma maior actividade desta enzima do que os opacos. Todos estes resultados indicam um papel da variação de fase na infecção pneumocócica e sublinham a importância deste fenómeno no ciclo de vida dos pneumococos. b) A elevada incidência de infecções invasivas em crianças com menos de dois anos deidade, bem como a maior frequência de resistência aos antimicrobianos, associada à rápida dispersão de alguns clones, levou à necessidade de uma monitorização contínua da epidemiologia de S. pneumoniae. A vacina conjugada 7-valente, recomendada para crianças com menos de dois anos de idade, foi introduzida em Portugal em 2001. O número limitado de serótipos presentes nesta vacina é expresso pela maioria das estirpes resistentes aos antimicrobianos. De modo a avaliar os efeitos causados pela implementação da vacina na estrutura genética da população pneumocócica causadora de infecções invasivas, determinaram-se os serótipos, a susceptibilidade aos antimicrobianos e a distribuição das linhagens genómicas das estirpes isoladas de 2004 a 2005.Os resultados foram comparados com os dados das estirpes isoladas entre 1999 e 2002 e com os dados disponíveis no laboratório referentes às estirpes isoladas no ano 2003. As alterações na estrutura genética da população pneumocócica ocorreram sobretudo desde2003. Tal como noutros países, após a introdução da vacina, verificou-se uma redução de casos de infecção invasiva causados pelos serótipos da vacina e um aumento de casos causados por serótipos menos comuns não cobertos pela vacina. Entre os dois períodos de estudo observou-se que os serótipos da vacina 4, 14 e 23F, diminuíram de frequência, enquanto que os serótipos não incluídos na vacina 3, 7F e 19A aumentaram. A vacinação levou à diminuição da potencial cobertura da vacina 7-valente de 63,2% (1999 a 2002) para apenas 28,6% (2004 e 2005), em crianças com menos de dois anos. Isto deveu-se, sobretudo, à prevalência do serótipo 19A que representava 37,1% das estirpes isoladas deste grupo etário entre 2004 e 2005. A potencial cobertura da vacina 23-valente permaneceu elevada, acima dos 80% em todos os grupos etários. Um efeito positivo foi a redução da resistência à penicilina entre os dois períodos de estudo.A não susceptibilidade à penicilina decresceu de 43,9% (1999 a 2002) para 30% (2004 e 2005), em crianças com idade < 6 anos, e atingiu 17,5% na totalidade das estirpes analisadas (2004 e 2005). Isto deveu-se à redução na prevalência dos serótipos 6B, 14 e 23F anteriormenteassociados com a não susceptibilidade à penicilina. Contudo, a resistência à tetraciclina aumentou de 7,1% para 12,8% e a resistência aos macrólidos aumentou de 9,4% para 15,1%, entre os dois períodos de estudo, na totalidade das estirpes analisadas. Em estirpes não susceptíveis à penicilina a multiresistência, relacionada com a resistência à tetraciclina e aos macrólidos, quase duplicou em crianças e adultos, atingindo valores próximos de 50%. O serótipo 19A passou a ser o mais frequente entre estirpes com o fenótipo MLSB e M e a resistência à tetraciclina apareceu pela primeira vez em estirpes com o fenótipo M isoladas em 2004 e 2005. O impacto da introdução da vacina conjugada em Portugal levou a uma alteração na composição clonal da população pneumocócica, caracterizada pelo aparecimento de 15 novas linhagens, desde 2003, e pelo aumento na prevalência de clones já estabelecidos expressando serótipos incluídos e não incluídos na vacina. Os resultados indicaram que fenómenos de troca capsular não foram frequentes pelo que a vacinação levou a uma menor importância de certos clones reconhecidos pela PMEN. Os clones Spain6B-2, Spain9V-3 (9V e 14), Colombia23F-26 e Spain23F-1 (19A) diminuíram de frequência entre 1999-2002 e 2004-2005. No entanto, estes clones continuam a agregar uma grande parte das estirpes resistentes aos antimicrobianos. Em conjunto, estes resultados sobre o impacto da implementação da vacina 7-valente em Portugal apontam para uma redução num curto prazo, dos potenciais benefícios da vacina.FCT (bolsa de doutoramento SFRH/BD/14158/2003

Absence of Synergism between a Dual-AMP Biogel and Antibiotics Used as Therapeutic Agents for Diabetic Foot Infections

Diabetic foot infections (DFIs) are frequently linked to diabetic-related morbidity and death because of the ineffectiveness of conventional antibiotics against multidrug-resistant bacteria. Pexiganan and nisin A are antimicrobial peptides (AMPs), and their application may complement conventional antibiotics in DFI treatment. A collagen 3D model, previously established to mimic a soft-tissue collagen matrix, was used to evaluate the antibacterial efficacy of a guar gum gel containing pexiganan and nisin alone and combined with three antimicrobials toward the biofilms of Staphylococcus aureus and Pseudomonas aeruginosa isolated from infected foot ulcers. Antimicrobials and bacterial diffusion were confirmed by spot-on-lawn and bacterial growth by bacterial count (cfu/mL). Our main conclusion was that the dual-AMP biogel combined with gentamicin, clindamycin, or vancomycin was not able to significantly reduce bacterial growth or eradicate S. aureus and P. aeruginosa DFI isolates. We further reported an antagonism between dual-AMP and dual-AMP combined with antibiotics against S. aureus.info:eu-repo/semantics/publishedVersio

The presence of the pilus locus is a clonal property among pneumococcal invasive isolates

<p>Abstract</p> <p>Background</p> <p>Pili were recently recognized in <it>Streptococcus pneumoniae </it>and implicated in the virulence of this bacterium, which led to the proposal of using these antigens in a future pneumococcal vaccine. However, pili were found to be encoded by the <it>rlrA </it>islet that was not universally distributed in the species. We examined the distribution of the pilus islet, using the presence of the <it>rlrA </it>gene as a marker for the locus, among a collection of invasive isolates recovered in Portugal and analyzed its association with capsular serotypes, clusters defined by the pulsed-field gel electrophoretic profiles (PFGE) and multilocus sequence types.</p> <p>Results</p> <p>Only a minority of the isolates were positive for the presence of the <it>rlrA </it>gene (27%). There was a high correspondence between the serotype and the presence or absence of <it>rlrA </it>(Wallace coefficient, W = 0.778). In particular, there was an association between the presence of <it>rlrA </it>and the vaccine serotypes 4, 6B, 9V and 14 whereas the gene was significantly absent from other serotypes, namely 1, 7F, 8, 12B and 23F, a group that included a vaccine serotype (23F) and serotype 1 associated with enhanced invasiveness. Even within serotypes, there was variation in the presence of the pilus islet between PFGE clones and a higher Wallace coefficient (W = 0.939) indicates that carriage of the islet is a clonal property of pneumococci. Analysis of <it>rlrA </it>negative isolates revealed heterogeneity in the genomic region downstream of the <it>rfl </it>gene, the region where the islet is found in other isolates, compatible with recent loss of the islet in some lineages.</p> <p>Conclusion</p> <p>The pilus islet is present in a minority of pneumococcal isolates recovered from human invasive infections and is therefore not an essential virulence factor in these infections. Carriage of the pilus islet is a clonal property of pneumococci that may vary between isolates expressing the same serotype and loss and acquisition of the islet may be ongoing.</p

Pseudomonads from wild free-living sea turtles in Principe Island, Gulf of Guinea

Dissemination of antibiotic resistance is a major concern, especially in aquatic environments, where pollution contributes for resistant bacteria selection. These strains may have serious health implications, especially for endangered species, including the sea turtles' hawksbill Eretmochelys imbricata and green turtles Chelonia mydas. We aimed to evaluate the presence of antibiotic resistant pseudomonads in wild sea turtles from Principe Island, Sao Tome and Principe, Guinea Gulf. Isolates were obtained from oral and cloacal swabs of free-living turtles by conventional techniques. Pseudomonads screening was performed by multiplex-PCR (oprl/oprL) and biochemical identification and antibiotic resistance profiling were achieved using Vitek2. All pseudomonad isolates were genotyped by Rep-PCR. Thirteen isolates were oprl-positive and classified as pseudomonads, eight from the genus Pseudomonas with the species P. aeruginosa, P. stutzeri, and P. mendocina, and five co-isolated Alcaligenes faecalis. The P. aeruginosa isolate was also oprL-positive. Regarding isolates susceptibility profile, 38.5% were susceptible to all antibiotics tested, and multidrug resistant (MDR) strains were not identified. DNA fingerprinting did not show any specific clonal-cluster similarity. Data on the worldwide incidence of antibiotic resistance among wildlife is still very scarce, especially concerning remote tropical areas. Since Pseudomonas genus has emerged as a group of increasingly reported opportunistic microorganisms in human and veterinary medicine with high resistance levels, it could be used as a tool for environmental resistance surveillance, particularly considering their ubiquity.Oceandrio de Lisboa, PortugalMarine Turtle Conservation Act - U.S. Fish & Wildlife Service grantInterdisciplinary Research Centre for Animal Health, Faculty of Veterinary Medicine, University of Lisbon (FMV/UL) [UID/CVT/00276/2013]Laboratory of Molecular and Cellular Technology, Queen Astrid Military Hospital, Brussels, Belgiuminfo:eu-repo/semantics/publishedVersio

Endothelium-mediated action of analogues of the endogenous neuropeptide kyotorphin (tyrosil-arginine) : mechanistic insights from permeation and effects on microcirculation

© 2016 American Chemical SocietyKyotorphin (KTP) is an endogenous peptide with analgesic properties when administered into the central nervous system (CNS). Its amidated form (l-Tyr-l-Arg-NH2; KTP-NH2) has improved analgesic efficacy after systemic administration, suggesting blood-brain barrier (BBB) crossing. KTP-NH2 also has anti-inflammatory action impacting on microcirculation. In this work, selected derivatives of KTP-NH2 were synthesized to improve lipophilicity and resistance to enzymatic degradation while introducing only minor changes in the chemical structure: N-terminal methylation and/or use of d amino acid residues. Intravital microscopy data show that KTP-NH2 having a d-Tyr residue, KTP-NH2-DL, efficiently decreases the number of leukocyte rolling in a murine model of inflammation induced by bacterial lipopolysaccharide (LPS): down to 46% after 30 min with 96 μM KTP-NH2-DL. The same molecule has lower ability to permeate membranes (relative permeability of 0.38) and no significant activity in a behavioral test which evaluates thermal nociception (hot-plate test). On the contrary, methylated isomers at 96 μM increase leukocyte rolling up to nearly 5-fold after 30 min, suggesting a proinflammatory activity. They have maximal ability to permeate membranes (relative permeability of 0.8) and induce long-lasting antinociception.FCT-MCTES is acknowledged for PhD fellowship SFRH/BD/52225/2013 to J.P. Marie Skłodowska-Curie Research and Innovation Staff Exchange (RISE) is acknowledged for funding: call H2020-MSCA-RISE-2014, Grant agreement 644167, 2015-2019.info:eu-repo/semantics/publishedVersio

The molecular epidemiology of multiple zoonotic origins of SARS-CoV-2

Understanding the circumstances that lead to pandemics is important for their prevention. Here, we analyze the genomic diversity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) early in the coronavirus disease 2019 (COVID-19) pandemic. We show that SARS-CoV-2 genomic diversity before February 2020 likely comprised only two distinct viral lineages, denoted A and B. Phylodynamic rooting methods, coupled with epidemic simulations, reveal that these lineages were the result of at least two separate cross-species transmission events into humans. The first zoonotic transmission likely involved lineage B viruses around 18 November 2019 (23 October–8 December), while the separate introduction of lineage A likely occurred within weeks of this event. These findings indicate that it is unlikely that SARS-CoV-2 circulated widely in humans prior to November 2019 and define the narrow window between when SARS-CoV-2 first jumped into humans and when the first cases of COVID-19 were reported. As with other coronaviruses, SARS-CoV-2 emergence likely resulted from multiple zoonotic events

First measurement of the Hubble Constant from a Dark Standard Siren using the Dark Energy Survey Galaxies and the LIGO/Virgo Binary–Black-hole Merger GW170814

International audienceWe present a multi-messenger measurement of the Hubble constant H 0 using the binary–black-hole merger GW170814 as a standard siren, combined with a photometric redshift catalog from the Dark Energy Survey (DES). The luminosity distance is obtained from the gravitational wave signal detected by the Laser Interferometer Gravitational-Wave Observatory (LIGO)/Virgo Collaboration (LVC) on 2017 August 14, and the redshift information is provided by the DES Year 3 data. Black hole mergers such as GW170814 are expected to lack bright electromagnetic emission to uniquely identify their host galaxies and build an object-by-object Hubble diagram. However, they are suitable for a statistical measurement, provided that a galaxy catalog of adequate depth and redshift completion is available. Here we present the first Hubble parameter measurement using a black hole merger. Our analysis results in , which is consistent with both SN Ia and cosmic microwave background measurements of the Hubble constant. The quoted 68% credible region comprises 60% of the uniform prior range [20, 140] km s−1 Mpc−1, and it depends on the assumed prior range. If we take a broader prior of [10, 220] km s−1 Mpc−1, we find (57% of the prior range). Although a weak constraint on the Hubble constant from a single event is expected using the dark siren method, a multifold increase in the LVC event rate is anticipated in the coming years and combinations of many sirens will lead to improved constraints on H 0

First Measurement of the Hubble Constant from a Dark Standard Siren using the Dark Energy Survey Galaxies and the LIGO/Virgo Binary-Black-hole Merger GW170814

We present a multi-messenger measurement of the Hubble constant H 0 using the binary–black-hole merger GW170814 as a standard siren, combined with a photometric redshift catalog from the Dark Energy Survey (DES). The luminosity distance is obtained from the gravitational wave signal detected by the Laser Interferometer Gravitational-Wave Observatory (LIGO)/Virgo Collaboration (LVC) on 2017 August 14, and the redshift information is provided by the DES Year 3 data. Black hole mergers such as GW170814 are expected to lack bright electromagnetic emission to uniquely identify their host galaxies and build an object-by-object Hubble diagram. However, they are suitable for a statistical measurement, provided that a galaxy catalog of adequate depth and redshift completion is available. Here we present the first Hubble parameter measurement using a black hole merger. Our analysis results in ${H}_{0}={75}_{-32}^{+40}\,\mathrm{km}\,{{\rm{s}}}^{-1}\,{\mathrm{Mpc}}^{-1}$, which is consistent with both SN Ia and cosmic microwave background measurements of the Hubble constant. The quoted 68% credible region comprises 60% of the uniform prior range [20, 140] km s−1 Mpc−1, and it depends on the assumed prior range. If we take a broader prior of [10, 220] km s−1 Mpc−1, we find {H}_{0 {78}_{-24}^{+96}\,\mathrm{km}\,{{\rm{s}}}^{-1}\,{\mathrm{Mpc}}^{-1} (57% of the prior range). Although a weak constraint on the Hubble constant from a single event is expected using the dark siren method, a multifold increase in the LVC event rate is anticipated in the coming years and combinations of many sirens will lead to improved constraints on H 0

Bacteriostatic and Antibiofilm Efficacy of a Nisin Z Solution against Co-Cultures of Staphylococcus aureus and Pseudomonas aeruginosa from Diabetic Foot Infections

Diabetes mellitus (DM) patients frequently develop diabetic foot ulcers (DFU) which are generally infected by a community of microorganisms, mainly Staphylococcus aureus and Pseudomonas aeruginosa. These bacteria exhibit a multi-drug resistance profile and biofilm-forming ability which represent a hurdle in the treatment of diabetic foot infections (DFI). We aimed to evaluate the potential of Nisin Z, an antimicrobial peptide (AMP), as an alternative treatment for severe DFI. Nisin Z shows antibacterial activity against Gram-positive and Gram-negative bacteria and an increased antibacterial effect against Gram-negatives when added to EDTA. As such, Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), Minimum Biofilm Inhibitory Concentration (MBIC), and Minimum Biofilm Eradication Concentration (MBEC) were determined for Nisin Z, Nisin Z + EDTA (0.4%), and Nisin Z + EDTA incorporated into guar gum, in order to test its efficacy against S. aureus and P. aeruginosa isolated from the same DFU. Results showed that Nisin Z added to the chelation agent EDTA displayed higher antibacterial and bacteriostatic efficacy against mono and dual co-cultures of S. aureus and P. aeruginosa, and higher antibiofilm efficiency against monocultures. Nisin Z was moderately cytotoxic at 200 &micro;g/mL. Prospect in vivo studies are needed to confirm the potential of Nisin Z supplemented with EDTA to be used as a complement to conventional antibiotic therapy for severe DFI

The Virtuous Galleria mellonella Model for Scientific Experimentation

The first research on the insect Galleria mellonella was published 85 years ago, and the larva is now widely used as a model to study infections caused by bacterial and fungal pathogens, for screening new antimicrobials, to study the adjacent immune response in co-infections or in host-pathogen interaction, as well as in a toxicity model. The immune system of the G. mellonella model shows remarkable similarities with mammals. Furthermore, results from G. mellonella correlate positively with mammalian models and with other invertebrate models. Unlike other invertebrate models, G. mellonella can withstand temperatures of 37 &deg;C, and its handling and experimental procedures are simpler. Despite having some disadvantages, G. mellonella is a virtuous in vivo model to be used in preclinical studies, as an intermediate model between in vitro and mammalian in vivo studies, and is a great example on how to apply the bioethics principle of the 3Rs (Replacement, Reduction, and Refinement) in animal experimentation. This review aims to discuss the progress of the G. mellonella model, highlighting the key aspects of its use, including experimental design considerations and the necessity to standardize them. A different score in the &ldquo;cocoon&rdquo; category included in the G. mellonella Health Index Scoring System is also proposed