Desenvolvimento de uma abordagem de ionização-dessorção a laser assistida por matriz acoplada a espetrometria de massa por tempo de voo para análise de biofilmes de Staphylococcus epidermidis formados in vitro

Dissertação de mestrado em BioengenhariaOs cateteres são a principal via de acesso venoso em pacientes que realizam hemodiálise (HD). Contudo, estes estão associados a elevadas taxas de mortalidade e morbilidade devido principalmente ao desenvolvimento de infeções. De facto, os cateteres facilitam o acesso ao sangue de microrganismos, em particular de Staphylococcus epidermidis, que possui a capacidade de aderir às superfícies do cateter e formar biofilme. O processo de formação de biofilme é multifatorial, podendo as proteínas intervenientes representar potenciais biomarcadores para o diagnóstico e monotorização terapêutica de infeções. Por conseguinte, o principal objetivo deste estudo consistiu na caracterização de biofilmes de S. epidermidis utilizando ionização/dessorção a laser assistida por matriz acoplada a espetrometria de massa por tempo de voo (MALDI-TOF MS). Inicialmente, foi realizada a pesquisa de microrganismos em cateteres de HD e investigada a capacidade de formação de biofilme in vitro de isolados clínicos de S. epidermidis. Adicionalmente, foi desenvolvida uma metodologia para a investigação do perfil proteico de sobrenadantes e das frações celulares (células de biofilme e planctónicas) de biofilmes de S. epidermidis formados in vitro, por MALDI-TOF MS. Este estudo revelou que a presença de microrganismos não é universal nos cateteres de HD. Contudo, S. epidermidis foi recuperado de cateteres e foi demonstrada a sua capacidade de formar biofilme in vitro. A análise por MALDI-TOF MS de sobrenadantes de biofilme mostrou que, nas condições testadas, não é possível obter espetros com qualidade, em termos do número de picos e de reprodutibilidade, invalidando a continuidade do seu estudo. Por outro lado, os estudos efetuados com frações celulares revelaram que os melhores espetros de MALDI-TOF MS, com maior número de picos detetados, se obtêm com: (1) a matriz ferulic acid (vs. a matriz α-cyano-4-hydroxycinnamic acid); (2) 2×108 UFC/posição da placa de MALDI-TOF MS (num intervalo de 2×106 a 6×108 UFC/posição); (3) com células intactas (vs. extratos celulares) lavadas e ressuspensas em ácido trifluoroacético. Apesar do método de MALDI-TOF MS ser rápido, simples e facilmente aplicável, a baixa reprodutibilidade observada neste estudo não permitiu a determinação de um perfil único, representativo e diferencial para cada fração celular. Em conclusão, este trabalho permitiu mostrar a importância da otimização de diferentes parâmetros experimentais nas análises de MALDI-TOF MS, sendo no entanto necessário o estudo de diferentes abordagens experimentais para a análise de biofilmes de S. epidermidis, antes de qualquer aplicação clínica desta técnica.Catheters are the most frequently used venous accesses in patients on hemodialysis (HD). However, these are associated with high rates of mortality and morbidity mainly due to the development of infections. In fact, catheters provide the access of microorganisms to the blood, particularly Staphylococcus epidermidis that has the ability to adhere to catheters surface and to form biofilms. The biofilm formation is a multifactorial process, and the proteins involved may represent potential biomarkers for diagnosis and therapeutic monitoring of infections. Therefore, the aim of this study was the characterization of S. epidermidis biofilms using matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF MS). Initially, the microbial population on HD catheters and the ability of clinical isolates of S. epidermidis to form biofilms in vitro was evaluated. Furthermore, it has been developed a methodology based on MALDI-TOF MS, to investigate the protein profile of supernatants and cellular fractions (biofilm and planktonic cells) of S. epidermidis biofilms formed in vitro. In this study, the analysis performed on catheters revealed that the presence of microorganisms is not universal in the HD catheters. However, S. epidermidis was recovered from catheters and it was demonstrated their ability to form biofilms in vitro. The MALDI-TOF MS analysis for biofilm supernatants showed that, under the tested conditions, it is not possible to obtain spectra with enough quality, regarding the number of peaks and the reproducibility, hampering their study. On the other hand, the studies concerning the cell fractions revealed that the best MALDI-TOF MS spectra, with higher number of peaks, are obtained with: (1) the ferulic acid matrix (vs. α-cyano-4-hydroxycinnamic acid matrix); (2) 2×108 CFU/MALDI-TOF MS plate position (in a range between 2×106 and 6×108 CFU/position); (3) with intact cells (vs. cell extracts) washed and resuspended in trifluoroacetic acid. Although the MALDI-TOF MS method was fast, simple and easily applicable, the low reproducibility observed in this study did not allow the determination of a unique, representative and differential profile for each cell fraction. In conclusion, this study revealed the importance of the optimization of several experimental parameters in MALDI-TOF MS analysis although it is necessary the study of different approaches for the analysis of S. epidermidis biofilms, before any clinical application of this technique

Conjugate vaccine serotypes persist as major causes of non-invasive pneumococcal pneumonia in Portugal despite declines in serotypes 3 and 19A (2012-2015)

Copyright: © 2018 Hora ́cio et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Non-invasive pneumococcal pneumonia (NIPP) is a frequent cause of morbidity and mortality worldwide. The 13-valent pneumococcal conjugate vaccine (PCV13) was included in the national immunization program of children living in Portugal in 2015. Until then, PCV7 (since late 2001) and PCV13 (since early 2010) were given through the private market. We determined the serotype distribution and antimicrobial susceptibility of isolates causing adult NIPP in 2012-2015 and compared the results with previously published data (2007-2011). There were 50 serotypes among the 1435 isolates. The most common were serotypes: 3 (14%), 11A (8%), 19F (6%), 23A (5%), 6C (5%), 19A (4%), 23B (4%), 9N (4%) and non-typable isolates (4%). When considering data since the availability of PCV13 for children in the private market, the proportion of PCV13 serotypes declined from 44.0% in 2010 to 29.7% in 2015 (p < 0.001), mainly due to early decreases in the proportions of serotypes 3 and 19A. In contrast, during the same period, PCV7 serotypes (11.9% in 2012-2015) and the serotypes exclusive of the 23-valent polysaccharide vaccine (26.0% in 2012-2015), remained relatively stable, while non-vaccine types increased from 27.0% in 2010 to 41.9% in 2015 (p<0.001). According to the Clinical and Laboratory Standards Institute (CLSI) breakpoints, penicillin non-susceptible and erythromycin resistant isolates accounted for 1% and 21.7%, respectively, of the isolates recovered in 2012-2015, with no significant changes seen since 2007. Comparison of NIPP serotypes with contemporary invasive disease serotypes identified associations of 19 serotypes with either disease presentation. The introduction of PCV13 in the national immunization program for children from 2015 onwards may lead to reductions in the proportion of NIPP due to vaccine serotypes but continued NIPP surveillance is essential due to a different serotype distribution from invasive disease.ANH was supported by a grant from Fundação para a Ciência e Tecnologia, Portugal SFRH/BD/81205/2011. This work was partly supported by Fundação para a Ciência e a Tecnologia, Portugal (PTDC/DTP-EPI/1555/2014), LISBOA-01-0145-FEDER-007391, project cofunded by FEDER, through POR Lisboa 2020 - Programa Operacional Regional de Lisboa, PORTUGAL 2020 and Fundação para a Ciência e a Tecnologia, and an unrestricted Investigator initiated project from Pfizer.info:eu-repo/semantics/publishedVersio

Conjugate vaccine serotypes persist as major causes of non-invasive pneumococcal pneumonia in Portugal despite declines in serotypes 3 and 19A (2012-2015).

Non-invasive pneumococcal pneumonia (NIPP) is a frequent cause of morbidity and mortality worldwide. The 13-valent pneumococcal conjugate vaccine (PCV13) was included in the national immunization program of children living in Portugal in 2015. Until then, PCV7 (since late 2001) and PCV13 (since early 2010) were given through the private market. We determined the serotype distribution and antimicrobial susceptibility of isolates causing adult NIPP in 2012-2015 and compared the results with previously published data (2007-2011). There were 50 serotypes among the 1435 isolates. The most common were serotypes: 3 (14%), 11A (8%), 19F (6%), 23A (5%), 6C (5%), 19A (4%), 23B (4%), 9N (4%) and non-typable isolates (4%). When considering data since the availability of PCV13 for children in the private market, the proportion of PCV13 serotypes declined from 44.0% in 2010 to 29.7% in 2015 (p < 0.001), mainly due to early decreases in the proportions of serotypes 3 and 19A. In contrast, during the same period, PCV7 serotypes (11.9% in 2012-2015) and the serotypes exclusive of the 23-valent polysaccharide vaccine (26.0% in 2012-2015), remained relatively stable, while non-vaccine types increased from 27.0% in 2010 to 41.9% in 2015 (p<0.001). According to the Clinical and Laboratory Standards Institute (CLSI) breakpoints, penicillin non-susceptible and erythromycin resistant isolates accounted for 1% and 21.7%, respectively, of the isolates recovered in 2012-2015, with no significant changes seen since 2007. Comparison of NIPP serotypes with contemporary invasive disease serotypes identified associations of 19 serotypes with either disease presentation. The introduction of PCV13 in the national immunization program for children from 2015 onwards may lead to reductions in the proportion of NIPP due to vaccine serotypes but continued NIPP surveillance is essential due to a different serotype distribution from invasive disease

Genomic information of strains SH4916 and SH5446

Description of the methods used to analyze the high-throughput sequencing information of strains SH4916 and SH5446. Annotated contigs of each of the strains are provided.<br