Scoping Review: The Empowerment of Alzheimer’s Disease Caregivers with mHealth Applications

Alzheimer’s Disease (AD) is one of the most prevalent neurodegenerative chronic diseases. As it progresses, patients become increasingly dependent, and their caregivers are burdened with the increasing demand for managing their care. Mobile health (mHealth) technology, such as smartphone applications, can support the need of these caregivers. This paper examines the published academic literature of mHealth applications that support the caregivers of AD patients. Following the PRISMA for scoping reviews, we searched published literature in five electronic databases between January 2014 and January 2021. Twelve articles were included in the final review. Six themes emerged based on the functionalities provided by the reviewed applications for caregivers. They are tracking, task management, monitoring, caregiver mental support, education, and caregiver communication platform. The review revealed that mHealth applications for AD patients’ caregivers are inadequate. There is an opportunity for industry, government, and academia to fill the unmet need of these caregiver

Streptococcus agalactiae causing human infections : genetic diversity and capsular switching

Tese de doutoramento, Ciências e Tecnologias da Saúde (Microbiologia), Universidade de Lisboa, Faculdade de Medicina, 2011Streptococcus agalactiae (group B streptococci, GBS) is primarily a colonizing agent of the genitourinary and gastrointestinal tracts of a significant proportion of the human population. It is, however, well established as a leading cause of bacterial sepsis and meningitis in neonates and is increasingly associated with invasive infections in adults. While vertical transmission is commonly accepted to be the cause of early-onset disease, the source of bacterial strains causing infection in the late-onset period is less well understood. Administration of intrapartum antimicrobial prophylaxis to colonized women has resulted in a striking decline in early-onset and maternal GBS disease, but late-onset infections have mostly remained unchanged. Moreover, antimicrobial prophylaxis raised concerns as to selection and emergence of GBS resistant strains and alternative prevention strategies have focused on the development of vaccines that hold promising, although still preliminary results. The aim of the work presented in this thesis was to characterize the population structure of GBS in Portugal, and to assess the genetic diversity of isolates recovered from vaginal colonization and invasive disease in different age groups, to contribute to the global epidemiology of GBS and our understanding of GBS population biology. To this end a set of common techniques was chosen, including serotyping, antimicrobial susceptibility testing, pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and surface protein gene profiling. In combination, these methods allowed the identification of the main genetic lineages circulating in Portugal and Barcelona, providing the means for an appropriate comparison of both. These studies started with the comparison of 64 isolates recovered from invasive infections in newborns in the Lisbon area with 269 isolates colonizing women in the third trimester of pregnancy, from the same period. The genetic lineages defined by both PFGE and MLST identified very diverse populations with reported differences in the prevalence of serotypes and clones in carriage and invasive disease. A major finding concerned the identification of an unusually high proportion of ST24 isolates among serotype Ia, further strengthened by the independent study of another population (212 neonatal isolates) from the Barcelona area. Despite the geographic distance, both studies from Barcelona and Lisbon revealed extensive similarity in terms of clonal structure and genetic lineages. The high prevalence in both the studies of a particular lineage serotype Ia, defined by ST24 and the surface protein gene bca, highlighted the importance of local dynamics, indicating that genetic evolution of GBS presents with a geographic structure and may depend on local factors. The subsequent analysis of 225 isolates recovered from non-pregnant adults in Portugal revealed a GBS population dominated by a more diverse clonal composition when compared to that of neonates, consistent with the broader spectrum of disease presentation in these patients and consequent multiplicity of genetic lineages. Invasive disease in this population increased with age and was more frequent among men. The dominance of serotype Ia in this population, regardless of age, highlighted the importance of this serotype in GBS pathogenesis as a leading cause of invasive infections in adults, not reported elsewhere but already noted among neonatal infections in the Iberian Peninsula. Furthermore, the high prevalence of ST24 in all these studies, as opposed to rare descriptions elsewhere, suggested that this lineage had enhanced invasiveness and was probably expanding as a regionally successful clone that may disseminate more globally. Macrolide resistance rates in Portugal did not show significant trends, even if macrolides have been used in intrapartum prophylaxis increasing the selective pressure on GBS. Macrolide resistance is disseminated in Portugal by both a multiclonal mechanism resulting from the spread of resistance genes throughout most serotypes and genetic backgrounds, as well as by clonal expansion of particular lineages, such as the serotype V ST1/alp3. One of the main purposes of the analysis of a significant number of GBS isolates was their classification into lineages sharing the same genetic background, which would allow the inference of genetic relationships between strains and their contextualization in the global epidemiology of GBS. However, the associations of phenotype-genotype or between different genetic traits were never absolute, highlighting the role of horizontal genetic transfer in the evolution of GBS. Capsular switching was anticipated to occur frequently within GBS, even though this species is not recognized to be naturally competent for the acquisition of foreign DNA. Substantial evidence provided by the epidemiological studies performed on the Portuguese GBS collections drove the search for capsular transformants within these populations. The results obtained confirmed the existence of capsular switching in GBS, but questioned the high frequency of these events estimated from previous studies. Serotyping errors probably justified the overrepresentation of capsular switching in epidemiological studies. The mechanism for these genetic transfer events involved the replacement of the whole capsular locus instead of the previously proposed genetic transfer of only the serotypespecific genes. Globally, the results presented in this thesis suggest that GBS has an apparent remarkably stable, both temporally and geographically, clonal structure. Against this, background diversification is ongoing and can depend on local factors. Capsular switching is likely contributing to diversification, however not as frequently as initially thought and may impact on the vaccine formulations currently under development. Despite increasing information on maternal colonization and invasive disease, a better understanding of colonization in adults and natural reservoirs of GBS is required for the appropriate management of the GBS infections.Streptococcus agalactiae (estreptococos do grupo B, GBS) é maioritariamente um agente colonizador dos tratos genito-urinário e gastrointestinal de uma proporção significativa da população humana. É, no entanto, reconhecido como uma das principais causas de sépsis e meningite bacteriana em recém-nascidos, e crescentemente associado a infecções invasivas em adultos. Enquanto a transmissão vertical é normalmente aceite como causa do aparecimento precoce da doença, a origem das estirpes bacterianas que causam infecção no período tardio é menos bem compreendida. A administração de profilaxia antimicrobiana intraparto a mulheres colonizadas resultou num declínio acentuado das infecções por GBS de início precoce e materna, mas as infecções de início tardio, na sua maioria, mantiveram-se inalteradas. Mais ainda, a profilaxia antimicrobiana levantou preocupações quanto à selecção e ao aparecimento de estirpes de GBS resistentes e as estratégias alternativas de prevenção têm-se focado no desenvolvimento de vacinas com resultados promissores, embora ainda preliminares. O objectivo do trabalho apresentado nesta tese foi caracterizar a estrutura da população de GBS em Portugal e avaliar a diversidade genética das estirpes isoladas de colonização vaginal e doença invasiva em diferentes faixas etárias, de forma a contribuir para a epidemiologia global de GBS e para a compreensão da biologia populacional de GBS. Para atingir esse objectivo foi escolhido um conjunto de técnicas, incluindo a serotipagem, testes de susceptibilidade a antimicrobianos, electroforese em gel de campo pulsado (PFGE), “multilocus sequence typing” (MLST) e determinação de perfis de proteínas de superfície. Em combinação, estes métodos permitiram a identificação das principais linhagens genéticas que circulam em Portugal e Barcelona, fornecendo meios para uma comparação apropriada de ambas. Estes estudos começaram com a comparação de 64 estirpes isoladas de infecções invasivas em recém-nascidos na região de Lisboa com 269 estirpes de colonização em mulheres no terceiro trimestre de gravidez, no mesmo período. As linhagens genéticas definidas por PFGE e MLST identificaram populações muito diversificadas, com diferenças descritas na prevalência dos serótipos e clones, em colonização e doença invasiva. Uma constatação importante diz respeito à identificação de um número excepcionalmente elevado de estirpes ST24 no serótipo Ia, reforçado pelo estudo independente de outra população (212 estirpes neonatais) da área de Barcelona. Apesar da distância geográfica, ambos os estudos de Barcelona e Lisboa revelaram uma enorme semelhança em termos da estrutura clonal e linhagens genéticas. A elevada prevalência em ambos os estudos de uma linhagem particular do serótipo Ia, definida pelo ST24 e pelo gene que codifica a proteína de superfície bca, salientaram a importância de dinâmicas locais, indicando que a evolução genética de GBS apresenta uma estrutura geográfica e pode depender de factores locais A análise subsequente de 225 estirpes isoladas de adultos, exceptuando grávidas, em Portugal revelou uma população de GBS dominada por uma composição clonal mais diversificada quando comparada com a de recém-nascidos, consistente com o espectro mais alargado de manifestações de doença nestes indivíduos e consequente multiplicidade de linhagens genéticas. A doença invasiva nesta população aumentou com a idade, sendo mais frequente entre os homens. A predominância do serótipo Ia nessa população, independentemente da idade, destacou a importância deste serótipo na patogénese da GBS como uma das principais causas de infecções invasivas em adultos, não relatada noutros locais mas já identificada nas infecções neonatais na Península Ibérica. Além disso, a elevada prevalência de ST24 em todos estes estudos, por oposição a raras descrições noutros locais, sugerem que esta linhagem tem maior capacidade invasiva e está provavelmente em expansão como um clone bem-sucedido a nível regional que pode disseminar mais globalmente. As taxas de resistência aos macrólidos em Portugal não apresentaram tendências significativas, ainda que os macrólidos sejam usados na profilaxia intraparto, aumentando a pressão selectiva sobre GBS. A resistência aos macrólidos está disseminada em Portugal, tanto por um mecanismo multiclonal que resulta na dispersão de genes de resistência na maior parte serótipos e clones, como pela expansão clonal de linhagens específicas, tais como a do serótipo V ST1/alp3. Um dos principais objectivos da análise de um número significativo de estirpes de GBS era a sua classificação em linhagens com o mesmo património genético, o que permitiria a inferência de relações genéticas entre estirpes, e a sua contextualização na epidemiologia global de GBS. Contudo, as associações fenótipo-genótipo ou entre características genéticas diferentes nunca foram absolutas, salientando o papel da transferência genética horizontal na evolução de GBS. Foi proposto que a transformação capsular ocorre frequentemente em GBS, apesar de esta espécie não ser reconhecida como naturalmente competente para a aquisição de DNA exógeno. Evidências substanciais fornecidas pelos estudos epidemiológicos realizados nas colecções portuguesas de GBS levaram à pesquisa de transformantes capsular nessas populações. Os resultados obtidos confirmaram a existência de transformação capsular em GBS, mas questionaram a elevada frequência destes eventos, estimada em estudos anteriores. Erros de serotipagem provavelmente justificam a elevada representação de transformação capsular em estudos epidemiológicos. O mecanismo na base destes eventos de transferência genética envolveu a substituição de todo o locus capsular em vez da transferência genética de apenas os genes específicos do serótipo, proposta anteriormente. Globalmente, os resultados apresentados nesta tese sugerem que os GBS possuem uma estrutura clonal, extremamente estável, tanto geográfica como temporalmente. Por oposição, a diversificação do património genético é permanente e pode depender de factores locais. A transformação capsular provavelmente contribui para esta diversificação, porém não tão frequentemente quanto inicialmente se pensava e pode ter um impacto na formulação das vacinas actualmente em desenvolvimento. Apesar do aumento da informação sobre a colonização materna e a doença invasiva, uma melhor compreensão da colonização em adultos e dos reservatórios naturais de GBS é necessária para uma abordagem mais fundamentada das infecções por GBS.Fundação para a Ciência e a Tecnologia (FCT, SFRH/BD/41761/2007) e programa POPH/FS

A novel multiplex PCR-based tool of typing strains of Staphylococcus aureus

PhD ThesisMethicillin resistant Staphylococcus aureus (MRSA) is an important nosocomial pathogen and morbidity and mortality rates associated with this pathogen have increased markedly in recent years. The prevalence of MRSA is no longer confined to hospital patients since MRSA infections have been increasingly reported in the community. More recently, community-acquired MRSA strains have become more prevalent and their infections are no longer confined to the community but have started to replace hospital-acquired MRSA in some health care settings. MRSA strains are generally resistant to several classes of antibiotics and are therefore difficult and costly to treat. Consequently, an understanding of the epidemiological characteristics of S. aureus is an essential tool for the management of its infections in both the hospital and community setting. The purpose of any epidemiology study, such as the investigation of an outbreak, is to identify the potential source(s) of an infection and to monitor their dissemination. The early identification of an outbreak, making use of a rapid, precise and simple MRSA typing technique, can lead to prompt and effective precautions that avoid further spread of the infection. Pulsed-field gel electrophoresis (PFGE) is considered the gold standard for MRSA typing and has been recently supported by multi-locus sequence typing (MLST). However, technical limitations restrict the use of PFGE and MLST in the majority of routine hospital laboratories: they are time-consuming, expensive, require specific expertise and specialist equipment. In this study a novel typing technique was developed for S. aureus, based on single nucleotide polymorphism (SNP) variations in and around SmaI-restriction sites (CCCGGG), following the analysis of eighteen S. aureus strains that have had their genomes sequenced. The developed SmaI-multiplex PCR genotyping method combines the high discriminatory power and reproducibility of PFGE, with the simplicity of a multiplex PCR-based technique that can be performed in a routine clinical laboratory. The validity of SmaI-multiplex PCR was carefully assessed in comparison with PFGE and MLST against many sequenced S. aureus strains and showed high discriminatory power and reproducibility. There was also high level of concordance in the clustering of strains analyzed by each of the techniques. The SmaI-multiplex PCR was ultimately evaluated against a large number of clinical S. aureus outbreak strains and was shown to be a useful tool for providing reliable epidemiological information for the investigation of clinical staphylococcal outbreaks. The newly developed technology is suitable for high throughput sample analysis, is relatively cheap and provides reliable and comparable genotyping data. At the same time, the SmaI-multiplex PCR meets most of the criteria of practical typing method: it is simple, inexpensive, highly discriminatory and does not require sophisticated equipment or expertise. Consequently, SmaI-multiplex PCR could be used routinely in any clinical microbiology laboratory since it relies on standard clinical laboratory apparatus (i.e. PCR machine and agarose gel electrophoresis). SmaI-multiplex PCR proved to be more discriminatory than MLST/SCCmec typing, but less discriminatory than PFGE. Currently the SmaI-multiplex PCR protocol takes between 4 to 6 hours; however, it would be possible to adapt this technology towards an automated genotyping assay using RT-multiplex PCR. This would reduce the processing time to less than 60 minutes. Since individual targets are identifiable on the basis of the size of their amplicons, the RT-PCR output could be processed directly via dedicated analytical software.The Ministry of Higher Education: King Abdul-Aziz University, Saudi Arabia

Human listeriosis : Grouping of human Listeria monocytogenes isolates with PFGE and AscI restriction enzyme

Isolates of Listeria monocytogenes (N=932) collected from human cases of invasive listeriosis in Sweden between 1958 and 2010 were serotyped and characterised with pulsed-field gelelectrophoresis (PFGE) and AscI restriction enzyme. The genotype diversity of L. monocytogenes isolates was investigated and related to genotypic results from epidemiological information on human infection, in order to detect possible clustering of L. monocytogenes genotypes over time, season, location, age, or gender (Paper I). From 1972 to 1995, serovar 4b was the predominant serovar; however, in 1996, serovar 1/2a became the major serovar among human listeriosis cases in Sweden. Based on the number and distribution of all bands in the profile, 63 PFGE types belonging to serovars 1/2b, 3b and 4b and 119 PFGE types belonging to serovars 1/2a and 1/2c were identified (Paper I). The PFGE types were further assembled into PFGE groups, based on the number and distribution of small bands below 145.5 kb (Papers II and III). As the genomic region of small bands is genetically more conservative than in large bands, the distribution of small bands establishes relatedness of strains and defines genetic markers for both lineages. Cold-smoked salmon (Salmo salar) and gravad salmon packed under modified atmosphere or vacuum from three manufacturers were purchased in Sweden and Germany in 2005 and the occurrence and levels of L. monocytogenes were analysed (Paper IV): 56 products were analysed and eleven harboured L. monocytogenes. From the positive samples, 56 isolates were analysed with AscI, and 11 isolates were further analysed with ApaI: five AscI PFGE types were identified, four belonging to serovar 1/2a and one to 4b. Forty-three (n=43: 76.8%) isolates shared serovar 1/2a and 13 (23.2%) shared serovar 4b and all AscI types were identified among human clinical strains in Sweden. Moreover, three gravad salmon samples harboured two PFGE types each from different lineages, serovar 1/2a and serovar 4b. Although, in most of the products, the level of L. monocytogenes was less than 100 cfu/g, the highest level was 1500 cfu/g. The occurrence of L. monocytogenes was 12.9% in gravad salmon, encountered in three manufacturers (A, B, C) and 28% in cold-smoked salmon only from manufacturer A. Although the level of L. monocytogenes in RTE fish products is generally low, these products, should be considered possible sources of listeriosis in Sweden. A patient may harbour more than one L. monocytogenes PFGE type that can be determined through PFGE and AscI restriction enzyme. However, to avoid misleading conclusions, several L. monocytogenes colonies should be isolated and characterised from different sites from the same patient or mother-baby pairs (Paper V).Karakterisering av Listeria monocytogenes-isolat från människa Listerios är en allvarlig men ovanlig sjukdom som orsakas av bakterien Listeria monocytogenes (L. m.). Det är framför allt personer med nedsatt immunförsvar som drabbas. Dit hör till exempel organtransplanterade, diabetiker, cancerpatienter, äldre människor, gravida kvinnor och nyfödda barn. De vanligaste symptomen är hjärnhinne-/hjärninflammation och blodförgiftning. Gravida kvinnor kan abortera. Dödligheten ligger på 20–30 % trots behandling. Man har på senare år sett en ökning av antalet listeriosfall i Sverige. Listerios är livsmedelsburen och riskprodukter är ätfärdiga livsmedel som är kylförvarade och förpackade i livsmedelsgas eller vakuum och äts kalla. Sådana är till exempel gravad/rökt fisk, dessertostar, smörgåsmat och färdiglagad mat som inte upphettas före förtäring. L. m. växer bra i kylskåp och hämmas inte av hållbarhetsbefrämjande förpackningsmetoder. För att få vetskap om hur vanligt förekommande L. m. är i kallrökt lax eller gravad lax (Salmo salar) förpackad i vakuum eller livsmedelsgas undersöktes 56 produkter inköpta i butiker i Sverige. I elva av dem påvisades L. m. av samma PFGE-typer som man tidigare sett hos sjuka människor. För att hitta lämpliga karakteriseringsmetoder som kan användas vid smittspårning har 932 L. m.-isolat härrörande från människor som insjuknat i invasiv listerios i Sverige under åren 1958–2010 undersökts närmare. Isolaten har serotypats samt också karakteriserats med den molekylärbiologiska metoden PFGE, där man studerar bakteriernas arvsmassa (DNA). Från 1972–1995 dominerade serotyp 4b, men från 1996 dominerade serotyp 1/2a. Avhandlingen visar att de ’’små banden’’ i varje isolats PFGE-profil kan användas för att dela in Listeria-bakterierna i olika huvudgrupper. I avhandlingen visas också att en listerios-patient kan vara infekterad med mer än en PFGE-typ. Vid smittspårning är det därför viktigt att karakterisera flera isolat från patienten och jämföra dessa med isolat från misstänkta livsmedel.

Epidemiological characterization, antimicrobial resistance and virulence mechanisms of human and animal streptococci

Dissertação para obtenção do Grau de Doutor em BiologiaStreptococcus agalactiae (Group B Streptococcus - GBS), Streptococcus dysgalactiae subsp. dysgalactiae (Group C Streptococcus - GCS) and Streptococcus uberis are relevant mastitis pathogens, a highly prevalent and costly disease in the dairy industry due to antibiotherapy and loss in milk production. However, molecular characterization of field isolates of Streptococcus spp. occurring in Portugal was not known prior to our studies and is important to improve therapeutic and disease control programs. The aims of this study were the identification of strain molecular features, and the evaluation of antimicrobial drug resistance patterns of S. agalactiae (n=60), S. dysgalactiae subsp. dysgalactiae (n=18) and S. uberis (n=30) collected from bovine subclinical mastitis between 2002/2003 in Portugal. Additionally, two S. dysgalactiae subsp. dysgalactiae strains associated with invasive disease(one collected from cattle and the other from a human), and six Streptococcus dysgalactiae subsp. equisimilis (group C or group G Streptococcus - GCS/GGS) strains from human infection were included in the study, for comparative purposes. Genotypic relationships were identified using pulsed-field gel electrophoresis (PFGE)/BioNumerics, S. agalactiae and S. uberis multi-locus sequence typing (MLST), macrolide and tetracycline resistance gene profiling, S. agalactiae molecular serotyping, virulence gene profiling, PCR-amplification for screening presence of specific genes and subsequent sequencing for phylogenetic analysis, and reverse transcriptase-PCR (RT-PCR) for gene expression analysis of selected genes. Also, a custom-designed microarray containing 220 virulence genes of the human pathogen Streptococcus pyogenes (Group A Streptococcus - GAS) was used to test bovine GCS S. dysgalactiae subsp. dysgalactiae and human GCS/GGS S.dysgalactiae subsp. equisimilis. Antimicrobial resistance was assessed by disk diffusion against penicillin, gentamicin, streptomycin, amoxicillin-clavulanic acid, cefazolin, cefoperazone,rifaximin, erythromycin, pirlimycin, tetracycline, vancomycin, chloramphenicol and the macrolide lincosamide resistance phenotypes (cMLSB, iMLSB, M, L). Among S. uberis three PFGE clonal groups (defined by at least 80% similarity) comprised almost half of total isolates, and 50% of GBS isolates were included in four major clonal groups (all farm-associated), which is indicative of a contagious route of transmission between animals. The occurrence of PFGE patterns sharing >82.8% and 100% similarity among S. dysgalactiae subsp. dysgalactiae isolates collected from different farms suggests an environmental source for this pathogen in our case. By MLST, we observed that all S. uberis sequence types (ST) were found to be novel (n=14), representing novel genomic backgrounds for this pathogen. Among GBS only three MLST lineages (ST-2, ST-23, and ST-61/ST-554) were detected revealing little heterogeneity among our bovine GBS collection. Five new cpsD-cpsE-cpsF sequences of the cps locus (encoding the capsular polysaccharide) were detected in >70% of the bovine GBS, which may represent new serotypes.Fundação para a Ciência e a Tecnologia - Doctoral Project (SFRH/BD/32513/2006

Insights into the dynamics of methicillin-resistant staphylococci in animals : a focus on Staphylococcus pseudintermedius in dogs

Tese especialmente elaborada para a obtenção do grau de Doutor em Ciências Veterinárias, especialidade de ClínicaStaphylococci are a group of bacteria with clinical, agricultural, and economic importance because of their wide range of virulence factors and ability to become resistant to antimicrobials. This thesis has pursued three main objectives: I. Determine the frequency of methicillin-resistant S. aureus (MRSA) strains in several animal species, identify the characteristics of strains present in animals and comparison with human strains MRSA nasal screening was performed in 71 horses and 307 calves, and the observed frequencies were 3% and 2%, respectively. Seventy-four MRSA isolated from 2001 to 2014 were characterized: fourteen spa types, three SCCmec types and three clonal complexes (CC) 5, CC22 and CC398, were found. Most isolates were multidrug-resistant. Fourteen MRSA CC398 strains had qac genes (13 qacG and 1 qacJ), while 4 isolates (three CC5 and one CC22) had insertions in the norA promoter gene. MRSA linages from pets (CC5 and CC22) harboured specific sets of virulence genes and a lower number of resistance genes than CC398 from livestock-animals. II. Reveal antimicrobial/biocide susceptibility patterns/trends and resistance genes in methicillin-resistant staphylococci (MRS) Several antimicrobial resistance patterns and genes were found in MRS from horses. Minimum bactericidal concentrations of biocides chlorhexidine acetate, benzalkonium chloride, triclosan and glutaraldehyde were lower than the recommended in-use concentrations for veterinary medicine, although two MRS carried plasmid-borne qacA and sh-fabI or qacB and qacH-like genes. An investigation on the evolution of resistance to 38 antimicrobials, corresponding mechanisms and molecular characteristics of 644 clinical Staphylococcus spp. isolates obtained from companion animals between 1999-2014 revealed resistance to the majority of antimicrobials and the number of mecA-positive strains increased significantly over time. Considering S. pseudintermedius, the methicillin-susceptible (MSSP) were genetically more diverse than methicillin-resistant (MRSP). All MRSP and two MSSP strains were multidrug- resistant, with several antimicrobial resistance genes identified. One MSSP isolate harbored a qacA and another a qacB gene. Three biocide products had high bactericidal activity (Otodine®, Clorexyderm Spot Gel®, Dermocanis Piocure-M®), while Skingel® failed to achieve a five log reduction in the bacterial counting. III. Study of the pathogenesis of S. pseudintermedius in dogs The agr type III predominated in MRSP. Five virulence genes were found in all strains and only spsO gene was significantly associated with MSSP. MSSP produced more biofilm on BHIB and BHIB+1% glucose than MRSP isolates. Several virulence genes encoding surface proteins and toxins were highly expressed in the MRSP strain (compared to MSSP). By whole proteome characterization of S. pseudintermedius through 2DE MALDI-TOF/TOF MS approach we were able to identify 367 unique proteins, of which 39 were surface proteins. By subsequent use of the serological proteome analysis (SERPA) approach we identified 4 antigenic proteins with promising features for vaccine development. These results indicate that MRS were widely disseminated in the studied animal population, the environment and people in contact with these animals. The resistant trends and mechanisms detected in MRS strains are worrying and make animals a reservoir of important MRS clones and genes. Biocides are still a good therapeutic choice, even in the presence of efflux genes. Higher expression of virulence genes may play a role in the rapid and widespread of MRSP clones. Dogs are able to mount an IgG-response against S. pseudintermedius and the proteins identified by the immune system can in the future be used as vaccine candidates.RESUMO - Estudo da dinâmica de estafilococos meticilina-resistente em animais – um foco no Staphylococcus pseudintermedius em cães - Os estafilococos são um grupo de bactérias com importância clínica, agrícola e económica devido à ampla gama de fatores de virulência e pela sua capacidade de se tornarem resistentes aos antimicrobianos. Esta tese debruçou-se sobre três objetivos principais: I. Determinar a frequência de estirpes S. aureus meticilina-resistente (MRSA) em diversas espécies animais, identificar as características das estirpes presentes em animais e comparar com estirpes humanas Colhemos zaragatoas de 71 cavalos e 307 vitelos para pesquisa de MRSA, e observaramse frequências de 3% e 2%, respetivamente. Foram caracterizadas setenta e quatro estirpes MRSA isoladas entre 2001-2014: catorze tipos de spa, três tipos de SCCmec e três complexos clonais (CC) 5, CC22 e CC398, foram encontrados. A maioria das estirpes (74%) eram multirresistentes. Catorze estirpes de MRSA CC398 tinha genes qac (13 qacG e 1 qacJ), enquanto 4 (três CC5 e um CC22) tinham inserções no gene promotor norA. As linhagens de MRSA de animais de estimação (CC5 e CC22) tinham conjuntos específicos de genes de virulência e um menor número de genes de resistência do que as linhagens associadas aos animais de produção (CC398). II. Revelar padrões/ tendências de suscetibilidade antimicrobiana/biocida e genes de resistência em estafilococos meticilina-resistente (MRS) Foram encontrados vários padrões e genes de resistência antimicrobiana em MRS de cavalos. As concentrações bactericidas mínimas dos biocidas acetato de clorhexidina, cloreto de benzalcónio, triclosan e glutaraldeído foram menores do que as recomendadas em medicina veterinária, embora dois MRS tivessem os genes plasmídicos qacA e sh-fabI ou qacB e um qacH-semelhante. Uma investigação sobre a evolução da resistência a 38 antimicrobianos, mecanismos correspondentes e características moleculares de 644 Staphylococcus spp. clínicos obtidos de animais de companhia entre 1999-2014 revelou resistência à maioria dos antimicrobianos. O número de estirpes mecA-positivo aumentou significativamente ao longo do tempo. Quanto aos S. pseudintermedius, os meticilina-suscetível (MSSP) eram geneticamente mais diversos do que os meticilina-resistente (MRSP). Todos os MRSP e 2 MSSP eram multirresistentes, com vários genes de resistência identificados. Um MSSP tinha um gene qacA e outro um qacB. Três produtos biocidas tinham elevada atividade bactericida (Otodine®, Clorexyderm Spot Gel®, Dermocanis Piocure-M®), enquanto Skingel® não conseguiu atingir uma redução de 5 log na contagem bacteriana. III. Estudo da patogenicidade de S. pseudintermedius em cães O tipo III agr predominou nos MRSP. Cinco genes de virulência foram encontrados em todas as estirpes e só o gene spsO foi significativamente associado com MSSP. MSSP produziu mais biofilme em BHIB e BHIB + 1% glucose que as estirpes de MRSP. Vários genes de virulência que codificam proteínas e toxinas de superfície foram altamente expressos na estirpe MRSP (em comparação com MSSP). Através da caracterização do proteoma total de S. pseudintermedius pela abordagem 2DE MALDI-TOF/TOF MS fomos capazes de identificar 367 proteínas únicas, das quais 39 eram proteínas de superfície. Posteriormente utilizámos a análise do proteoma serológico (SERPA) que identificou quatro proteínas antigénicas com características promissoras para o desenvolvimento de vacinas. Estes resultados indicam que MRS estavam amplamente disseminados na população animal estudada, no ambiente e nas pessoas em contato com esses animais. As tendências de resistência e os mecanismos detetados em estirpes MRS são preocupantes tornando os animais um reservatório de clones MRS e genes. Os biocidas ainda são uma boa opção terapêutica, mesmo na presença de bombas de efluxo. Uma maior expressão de genes de virulência pode desempenhar um papel na rápida expansão de clones de MRSP. Os cães foram capazes de montar uma resposta IgG contra S. pseudintermedius e as proteínas identificadas pelo sistema imunológico podem, no futuro, ser utilizadas como candidatos vacinais

Testimonial of Professional Contributions in the Biotechnology Field (1991-2019). Pulmonary Infectious Agents Streptococcus pneumoniae and Mycobacterium tuberculosis

Relatório elaborado nos termos do Despacho n.º 20/2010 para obtenção do Grau de Mestre em Biotecnologia por Licenciados “Pré-Bolonha”Pulmonary infectious diseases, like pneumonia and tuberculosis, have been pervasive throughout the world for centuries; they kill millions of people annually worldwide and pose an increased threat when associated with antibiotic resistance and viral co-infections such as influenza and, more recently, Covid-19. This professional activity report summarizes some of my work in the last three decades, using biotechnology to type, characterize or detect two major bacterial infectious agents, Streptococcus pneumoniae and Mycobacterium tuberculosis. Strain typing techniques, like Pulsed Field Gel Electrophoresis and Penicillin Binding Protein patterns, were used to identify Streptococcus pneumoniae clones from different geographic areas in the early-1990s, showing strong evidence of clonal dissemination locally and across the globe, from Spain to Iceland and to the United States. In the mid-1990s, an in vivo expression technology system based on a promoter-trap was constructed to enable the identification of Mycobacterium tuberculosis genes upregulated in human macrophages, through selection for antibiotic resistance. This work led to the identification of eight genes likely relevant for Mycobacterium tuberculosis virulence. Several were later confirmed to be required for infection or survival in the host. A single-step molecular diagnostic real-time PCR assay for the detection of multidrug resistant Mycobacterium tuberculosis was commercialized in the late 2000s, with great impact in the detection of tuberculosis, especially in developing countries. The mechanism of the assay is presented here as published by its inventors, as well as with my role in the industry, from the development of processes for production and quality control, to regulation-compliant distribution, troubleshooting and development of improvements.As doenças infeciosas pulmonares, como a pneumonia e a tuberculose, têm tido, durante séculos uma prevalência mundial significativa, matando milhões de pessoas anualmente e representando uma ameaça crescente quando associadas à resistência a antibióticos ou coinfeções virais, como a gripe e, recentemente, o Covid-19. Este relatório de atividade profissional resume parte do meu trabalho nas últimas três décadas, usando técnicas de biotecnologia para identificar, caracterizar ou detetar dois importantes agentes infeciosos de origem bacteriana, Streptococcus pneumoniae e Mycobacterium tuberculosis. Técnicas de identificação de estirpes, como eletroforese tipo Pulsed Field e padrões de proteínas com afinidade à penicilina, foram usadas para identificar clones de Streptococcus pneumoniae de diferentes áreas geográficas no início da década de 90, mostrando forte evidência de disseminação, não só a nível local, mas também através do globo, nomeadamente, de Espanha para a Islândia e Estados Unidos. Em meados da década de 90, um sistema de expressão in vivo foi construído para permitir a identificação de genes de Mycobacterium tuberculosis com expressão elevada em macrófagos humanos, através de seleção por resistência a antibióticos. Este trabalho levou à identificação de oito genes com probabilidade de serem relevantes para a virulência de Mycobacterium tuberculosis; vários posteriormente confirmados como necessários para a infeção ou sobrevivência no hospedeiro. Mais recentemente, foi comercializado um teste de real-time PCR para diagnóstico molecular de Mycobacterium tuberculosis com multirresistência a antibióticos, com grande impacto na deteção da tuberculose, principalmente em certos países em desenvolvimento. O mecanismo de deteção é apresentado aqui conforme publicado pelos seus inventores, bem como o meu papel na indústria de diagnósticos moleculares, desde o desenvolvimento de processos de produção e controle de qualidade, até a distribuição em conformidade com os respetivos regulamentos, passando por resolução de problemas e desenvolvimento de melhorias

Examining antibiotic resistance in the feedlot cattle industry using real-time, quantitative PCR (qPCR) and enterococci as an indicator bacterium

Antibiotics are administered to livestock at subtherapeutic levels to maintain animal health. Many of the antibiotics used are analogues or the same as those used in human medicine, raising the possibility that genes conferring resistance arise within agricultural production systems. This thesis examined antibiotic resistance in the Canadian beef feedlot industry. Real-time, quantitative PCR was used to examine differences in the relative abundance of eighteen resistance genes across five antibiotic families from feedlot cattle faeces and urban environments. The effect of infeed administration and withdrawal of tylosin phosphate on macrolide resistance was examined using enterococci as an indicator bacterium. Resistant enterococci (n=21) were selected for whole-genome sequencing and comparative genomics. Results presented here show that the relative abundance of resistance genes differs between cattle feedlots and urban environments, likely a reflection of differences in antibiotic use. Sulfonamide, fluoroquinolone and β-lactam resistance genes predominated in urban wastewater, whilst tetracycline resistance genes were more prevalent in cattle faeces. Macrolide use in cattle production increased the proportion of erythromycinand tylosin-resistant enterococci. However, withdrawal of tylosin from the diet appeared to contribute to reduced macrolide resistance in enterococci. Comparative genomics revealed resistance to macrolides was present on mobile genetic elements, specifically the Tn917 transposon harbouring erm(B). This transposon was identified in both Enterococcus hirae and Enterococcus faecium suggesting inter-species transfer of resistance genes may occur in the bovine gastrointestinal tract. Furthermore, the integrative conjugative elements (ICEs) Tn916 and Tn5801, both conferring tetracycline resistance, were identified in E. faecium. As the cost of genomic sequencing continues to decrease, further investigation of ICEs using whole genome sequencing will help determine if there are linkages between enterococci isolates from bovine environmental and human clinical sources and whether bovine enterococci represent a source to the dissemination and spread of antibiotic resistance

Update on the population structure of MRSA causing infection in a central hospital and in healthcare centers in Portugal

RESUMO:Staphylococcus aureus é um dos principais agentes patogénicos humanos, sendo frequentemente associado a infecções nosocomiais e infecções na comunidade. A prevalência de S. aureus resistentes à meticilina (MRSA) em hospitais portugueses é uma das mais elevadas da Europa e tem sido caracterizada extensivamente; contrariamente, a prevalência e epidemiologia de MRSA na comunidade em Portugal não tem sido devidamente seguida. Com o objectivo de compreender as causas possíveis do aumento na frequência de MRSA num dos maiores hospitais centrais portugueses (HSM) ao longo de 17 anos, isolados de MRSA recolhidos em 1993 (n=54) e 2010 (n=180) de pus, sangue e urina foram analisados por PFGE, MLST, tipagem do spa e tipagem de SCCmec. Os resultados mostraram que ocorreu uma mudança global nos tipos clonais predominantes, onde o clone ST22-IVh substituiu os clones, ST239-IIIvar e ST247-I, representando mais de 70% da população actual. Além disso, entre 1993 e 2010 verificou-se um aumento na diversidade genética dos tipos clonais de MRSA. Para determinar a frequência e a natureza clonal de MRSA e S. aureus sensíveis à meticilina (MSSA) isolados de infecções de pele e tecidos moles (SSTI) em pessoas que frequentam centros de saúde em Portugal, 73 amostras foram recolhidas em nove centros de saúde (Rede Médicos Sentinela). Isolou-se um total de 40 S. aureus (55%), dos quais 17,5% eram MRSA. Os isolados de MRSA pertenciam aos clones ST22-IVh (n=4), ST5-IVc (n=2) e ST105-II (n=1), que foram descritos neste estudo como sendo clones de origem hospitalar. Os nossos resultados sugerem que o aumento da frequência de MRSA no HSM pode estar associado à emergência de um clone de MRSA com maior capacidade epidémica. Além disso, verificámos que a principal causa de SSTI em pessoas que frequentam centros de saúde em Portugal são MRSA de origem hospitalar e não MRSA associados à comunidade.------ABSTRACT: Staphylococcus aureus is one of the most important human pathogens, being a major cause of infections worldwide both in the hospital and in the community. In Portugal, the prevalence of methicillin resistant S. aureus (MRSA) in hospitals is one of the highest in Europe and has been characterized extensively; contrarily the prevalence and epidemiology of MRSA in the community has not been followed in a meaningful way. To understand the epidemiological events that could explain a steep increase in MRSA frequency in a major Portuguese central hospital (HSM) within a 17 year period, two MRSA collections recovered in 1993 (n=54) and 2010 (n=180) from pus, blood and urine were analyzed by PFGE, MLST, spa and SCCmec typing. The results showed that a major clonal shift occurred, wherein ST22-IVh clone has replaced the previous ST239-IIIvar and ST247-I clones and accounts for more than 70% of the present population. Moreover, an increase in genetic diversity of MRSA clonal types was observed between the two study periods. With the aim of determining the frequency and clonal nature of MRSA and methicillin-susceptible S. aureus (MSSA) causing skin and soft tissue infections (SSTI) in patients attending healthcare centers in Portugal, 73 samples were collected from nine healthcare centers (Medicos Sentinela Network). A total of 40 S. aureus were isolated, accounting for 55% of the SSTI, of which 17.5% were MRSA. MRSA isolates belonged to ST22-IVh (n=4), ST5-IVc (n=2) and ST105-II (n=1) that have also been described in the hospital in an equivalent period. Our results suggest that the increase in MRSA frequency in HSM may be associated to the emergence of a MRSA clone with higher epidemic potential. Moreover, we propose that the spillover of MRSA from the hospital rather than community-associated-MRSA was the main cause of SSTI in persons attending healthcare centers in Portugal