Virulence characterization and antimicrobial resistance of major bacterial genera from diabetic foot infections

Tese de Doutoramento em Ciências Veterinárias na especialidade de Ciências Biológicas e BiomédicasDiabetes mellitus is a major chronic disease that continues to increase significantly. One of the most important and costly complications of diabetes is the development of foot ulcers, colonized by pathogenic and antimicrobial resistant bacteria, which may be responsible for impairing its successful treatment. Diabetic foot ulcer (DFU) bacterial communities can be organized in polymicrobial biofilms, which may be responsible for its chronicity. The ability of these communities to produce biofilm was evaluated and was higher when compared to biofilm formation by individual species. Staphylococcus aureus is one of the most prevalent species in diabetic foot infections (DFI). Staphylococci isolated from DFU in patients from the Lisbon area were identified, genotyped and screened for virulence and antimicrobial resistance traits. The isolates showed high genomic diversity, were resistant to important clinically antibiotics and expressed relevant virulence determinants. As biofilm formation is one of the most important virulence traits of S. aureus, the antimicrobial susceptibility patterns of biofilm-producing S. aureus strains were also analysed. The minimum biofilm inhibitory and eradication concentrations were determined for ten antimicrobial compounds. Staphylococci biofilms were resistant to antibiotic concentrations ten to thousand times higher than those effective for planktonic cells. Furthermore, the enterococci frequently isolated from DFI, were also identified and characterized, showing high antimicrobial resistance and important virulence traits. Since DFI are often caused by resistant bacteria, it is necessary to find alternatives to antibiotic therapy, such as phage therapy. The inhibitory potential of five bacteriophages, previously characterized, was evaluated against established biofilms formed by S. aureus, P. aeruginosa and A. baumannii. A significant cell reduction after phage exposure was observed, mainly after multiple treatments. DFI are very complex and studies on this topic are scarce. It is necessary to intensify research in order to develop more adequate therapeutic protocols for this type of infection.RESUMO - Caracterização da virulência e resistência a antimicrobianos dos principais géneros bacterianos envolvidos em infeções de pé diabético - Diabetes mellitus é uma doença crónica com grande impacto em saúde pública e cuja incidência continua a aumentar significativamente em todo o mundo, atingindo atualmente mais de 400 milhões de pessoas. Uma das complicações mais importantes da diabetes e associada a gastos económicos significativos são as úlceras de pé diabético. Uma vez que a camada protetora de pele é danificada, os tecidos profundos ficam expostos à infeção bacteriana, a qual pode evoluir rapidamente. As infeções das úlceras de pé diabético são a causa mais comum de internamento hospitalar de pacientes diabéticos e uma importante causa de morbilidade, levando frequentemente à amputação dos membros inferiores. Estas infeções podem ser promovidas por bactérias potencialmente patogénicas e resistentes aos compostos antimicrobianos, prejudicando assim o sucesso do tratamento. As comunidades bacterianas presentes nas úlceras podem estar organizadas em biofilmes polimicrobianos, que contribuem para que as infeções se tornem crónicas e muito difíceis de resolver. Foi avaliada a capacidade de produção de biofilme por comunidades polimicrobianas de isolados bacterianos de pé diabético, utilizando um ensaio de microtitulação em placa com “Alamar Blue” (AB) e uma técnica de Hibridação In Situ Fluorescente Múltipla (MFISH). Esta avaliação foi realizada em três períodos de incubação distintos (24, 48 e 72 horas), depois da determinação da capacidade de formação de biofilme por 95 isolados de úlceras de pé diabético pertencentes a vários géneros bacterianos (Staphylococcus, Corynebacterium, Enterococcus, Pseudomonas e Acinetobacter). Todos os isolados apresentaram a capacidade de produzir biofilme às 24 horas, sendo que a quantidade de biofilme produzido aumentou com o tempo de incubação. Pseudomonas apresentou a capacidade mais elevada de produção de biofilme, seguida de Corynebacterium, Acinetobacter, Staphylococcus e por fim, Enterococcus. Foram encontradas diferenças estatisticamente significativas na capacidade de formação de biofilme entre os três períodos de incubação. As comunidades polimicrobianas produziram mais biofilme do que as espécies individualmente. As comunidades formadas por Pseudomonas + Enterococcus, Staphylococcus + Acinetobacter e Corynebacterium + Staphylococcus formaram mais biofilme do que as comunidades formadas por Enterococcus + Staphylococcus e por Enterococcus + Corynebacterium. O comportamento biológico das diferentes espécies bacterianas nos biofilmes polimicrobianos tem implicações clínicas muito importantes para o sucesso do tratamento deste tipo de infeções. A sinergia entre as bactérias presentes em biofilmes multiespécies foi descrita previamente, sendo que este trabalho representa o primeiro estudo sobre a evolução temporal da formação de biofilme por parte de comunidades polimicrobianas isoladas de úlceras de pé diabético, incluindo várias espécies. [...]Centro de Investigação Interdisciplinar em Sanidade Animal” (CIISA) of Faculty of Veterinary Medicine, University of Lisbon, PortugalN/

In vitro design of a novel lytic bacteriophage cocktail with therapeutic potential against organisms causing diabetic foot infections

In patients with diabetes mellitus, foot infections pose a significant risk. These are complex infections commonly caused by Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii, all of which are potentially susceptible to bacteriophages. Here, we characterized five bacteriophages that we had determined previously to have antimicrobial and wound-healing potential in chronic S. aureus, P. aeruginosa and A. baumannii infections. Morphological and genetic features indicated that the bacteriophages were lytic members of the family Myoviridae or Podoviridae and did not harbour any known bacterial virulence genes. Combinations of the bacteriophages had broad host ranges for the different target bacterial species. The activity of the bacteriophages against planktonic cells revealed effective, early killing at 4 h, followed by bacterial regrowth to pre-treatment levels by 24 h. Using metabolic activity as a measure of cell viability within established biofilms, we found significant cell impairment following bacteriophage exposure. Repeated treatment every 4 h caused a further decrease in cell activity. The greatest effects on both planktonic and biofilm cells occurred at a bacteriophage : bacterium input multiplicity of 10. These studies on both planktonic cells and established biofilms allowed us to better evaluate the effects of a high input multiplicity and a multiple-dose treatment protocol, and the findings support further clinical development of bacteriophage therapy.supported by TechnoPhage S.A. and Tecnifar; Foundation for Science and Technology (PTDC/SAUMIC/122816/2010 – ‘Biofilms in diabetic foot: microbial virulence characterization and cross-talk of major isolates’); FCT PhD fellowship (SFRH/BD/72872/2010); FCT ‘Ciência 2007’ programmeinfo:eu-repo/semantics/publishedVersio

Relatório estágio profissional

Relatório final do estágio profissionalizante do 6.º an

Systematic Literature Review of Open Infrastructure BIM

Representation and modeling using the building information modeling (BIM) methodology of civil works have become the subject of increasing attention in recent years, thanks to the potential offered by Open Infrastructure BIM (I-BIM). However, the complexity of infrastructure works, i.e., the variety of construction and technological systems, makes Open I-BIM very complex and challenging. The lack of systemic knowledge on the subject is another challenging factor. The aim of the following research work is to provide a synoptic overview of the existing scientific research, accompanied by the most recent studies in the field of computer modeling, its applications, and the main opportunities that Open I-BIM offers to the infrastructure sector. After a thorough review of 198 scientific articles published between 2013 and 2023, this study systematically presents a holistic review and critical reflection on the current status of the use of Open BIM in the infrastructure sector, with a focus on the development of the tools and methods used. The outcome of this work constitutes a systematic review of the literature with a bibliometric analysis on Open I-BIM, which is able to provide a knowledge base for identifying research trends, common problems, and the potential of developed methods

Arcobacter species detection in Italian composite foods

Considering that composite foods are frequently involved in the transmission of foodborne diseases and given the growing public health interest around the emerging genus Arcobacter, the aim of the present study was to assess the occurrence of Arcobacter spp. carrying virulence-associated genes in composite foods, in order to contribute to outlining the specific risks posed by emerging pathogens. Overall, Arcobacter spp. were detected in 18/75 (24%) samples. Biomolecular assays revealed A. butzleri in 36/54 (67%) and Arcobacter spp. in 18/54 (33%) isolates. PCRs aimed at the nine putative virulence genes demonstrated widespread distribution of these genes among A. butzleri isolates, while Arcobacter spp. isolates harboured only ciaB, mviN and hecA genes. This study provides more information regarding the health risks associated with the consumption of composite foods, underlying their role as a potential source of human Arcobacter infection

Polymicrobial biofilms by diabetic foot clinical isolates

© Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2015Diabetes mellitus is a major chronic disease that continues to increase significantly. One of the most important and costly complications of diabetes is foot ulceration that may be colonized by pathogenic and antimicrobial resistant bacteria, which may express several virulence factors that could impair treatment success. These bacterial communities can be organized in polymicrobial biofilms, which may be responsible for diabetic foot ulcer (DFU) chronicity. We evaluated the influence of polymicrobial communities in the ability of DFU isolates to produce biofilm, using a microtiter plate assay and a multiplex fluorescent in situ hybridization, at three time points (24, 48, 72 h), after evaluating biofilm formation by 95 DFU isolates belonging to several bacterial genera (Staphylococcus, Corynebacterium, Enterococcus, Pseudomonas and Acinetobacter). All isolates were biofilm-positive at 24 h, and the amount of biofilm produced increased with incubation time. Pseudomonas presented the higher biofilm production, followed by Corynebacterium, Acinetobacter, Staphylococcus and Enterococcus. Significant differences were found in biofilm formation between the three time points. Polymicrobial communities produced higher biofilm values than individual species. Pseudomonas + Enterococcus, Acinetobacter + Staphylococcus and Corynebacterium + Staphylococcus produced higher biofilm than the ones formed by E. faecalis + Staphylococcus and E. faecalis + Corynebacterium. Synergy between bacteria present in dual or multispecies biofilms has been described, and this work represents the first report on time course of biofilm formation by polymicrobial communities from DFUs including several species. The biological behavior of different bacterial species in polymicrobial biofilms has important clinical implications for the successful treatment of these infections.This study was conducted with the financial support of the Foundation for Science and Technology (FCT Project PTDC/SAU-MIC/122816/2010-Biofilms in diabetic foot: microbial virulence characterization and cross-talk of major isolates). Carla Mottola holds a PhD fellowship (SFRH/BD/72872/2010) from FCT, Portugalinfo:eu-repo/semantics/publishedVersio

Characterization of multidrug-resistant diabetic foot ulcer enterococci

Introduction: Diabetes mellitus is a highly prevalent chronic progressive disease with complications that include diabetic-foot ulcers. Methods: Enterococci isolated from diabetic-foot infections were identified, evaluated by macrorestriction analysis, and screened for virulence traits and antimicrobial resistance. Results: All isolates were considered multidrug-resistant, cytolysin and gelatinase producers, and the majority also demonstrated the ability to produce biofilms. Conclusions: These results indicate the importance of enterococci in diabetic-foot infection development and persistence, especially regarding their biofilm-forming ability and resistance to clinically relevant antibiotics.info:eu-repo/semantics/publishedVersio

Presenza e caratterizzazione di Arcobacter spp. in ortaggi di IV gamma prodotti in Italia meridionale

Le informazioni disponibili in letteratura relative alla presenza di Arcobacter spp. negli alimenti di origine vegetale sono attualmente carenti. Ampiamente documentato è, tuttavia, il ruolo assunto da tali alimenti nel determinismo di malattie a trasmissione alimentare. Obiettivo del presente studio è, pertanto, acquisire dati relativi alla presenza di Arcobacter spp. negli ortaggi ready-to-eat (RTE), evidenziare specifici tratti di virulenza e genotipizzare gli isolati, per implementare e attuare una mirata valutazione e relativa gestione del rischio microbiologico. Nell'ambito del presente lavoro sono stati considerati ortaggi RTE e, specificatamente, 56 campioni di lattuga e 54 campioni di rucola, provenienti da uno stabilimento di ortaggi di IV gamma sito nella regione Puglia. Il percorso sperimentale ha previsto, inizialmente, l’isolamento colturale di Arcobacter spp., effettuato su terreno solido specifico. Le colonie, isolate come appartenenti presumibilmente ad Arcobacter spp., sono state, quindi, identificate tramite Multiplex-PCR e sequenziamento del gene rpob. Gli isolati, successivamente, sono stati caratterizzati in base alla presenza di specifici fattori di virulenza e, infine, genotipizzati mediante Multi-Locus Sequence Type (MLST). L’isolamento colturale ha rilevato la presenza di Arcobacter spp. in 16/110 (14.5 %) ortaggi RTE e, in particolar modo, in 11 campioni di lattuga e 5 campioni di rucola. Dall'analisi biomolecolare effettuata sui 16 isolati, 15 sono risultati appartenere alla specie A. butzleri e 1 alla specie A. cryaerophilus. Sono stati inoltre rilevati specifici tratti di patogenicità diversamente distribuiti nei 16 isolati. La genotipizzazione eseguita mediante MLST ha evidenziato la presenza di soltanto 6 Sequence Type (STs) e, quindi, una esigua variabilità genetica, attribuibile probabilmente all'origine univoca dei campioni considerati. Lo studio conferma la presenza di Arcobacter spp. negli ortaggi RTE, imputabile principalmente alla contaminazione della materia prima e sollecita l’applicazione di appropriate e corrette pratiche agronomiche. L’impiego di fertilizzanti, quali letame e liquame non correttamente trattati, infatti, è la principale causa di contaminazione microbiologica dei vegetali. Inoltre l'individuazione di specifici fattori di virulenza e la genotipizzazione molecolare di A. butzleri e A. cryaerophilus, possibili responsabili di gastroenterite ad eziologia ignota, evidenziano la necessità di introdurre un sistema di sorveglianza basato sulla caratterizzazione biomolecolare per una integrata valutazione del rischio microbiologico degli ortaggi RTE, per implementare un innovativo controllo della filiera e, conseguentemente, degli alimenti composti