Análise genotípica de isolados de Mycobacterium tuberculosis de um hospital em Lisboa, Portugal

AbstractPortugal has one of the highest tuberculosis notification rates of the European Union with Lisbon Health Region having an incidence rate well above the national average. The present study analyses the transmission, drug susceptibility and characteristics of a study population from a Central Lisbon’s Hospital. One hundred and thirty-two Mycobacterium tuberculosis clinical isolates were previously tested for drug susceptibility to first-line drugs. The multidrug (MDR) resistance rate was found to be 3.0%, while 13.6% of the isolates were resistant to one or more first-line drugs. HIV serology was available for 98 patients, 26 (26.5%) were positive. Genotyping was performed by MIRU-VNTR and 53 (40,2%) out of the 132 isolates were found to be distributed through 17 MIRU-VNTR clusters of two or more isolates. Lisboa strains accounted for 25.8% of all strains. We conclude that transmission of resistant and susceptible Mycobacterium tuberculosis strains is occurring, with special concern for Lisboa strains.Rev Port Pneumol 2009; XV (5): 761-76

Interspecies interactions and bacteriophage control in S. Enteritidis and E. coli mixed biofilms

Microbiotec'17 - Congress of Microbiology and Biotechnology 2017Background Salmonella Enteritidis and Escherichia coli are both important foodborne pathogens, commonly related to outbreaks1. Their ability to form biofilms contributes to their virulence and enables their survival on different food contact surfaces2. In mixed biofilms, interspecies interactions can occur, resulting in positive, negative or neutral outcomes to each species3,4. As these microbial communities present great resistance to antimicrobials used in food industries (disinfectants, biocide, antimicrobials), bacteriophages, bacterial viruses, can be regarded as good and safe candidates for biofilm biocontrol5. This study aimed at characterizing the interactions established between two S. Enteritidis and two E. coli strains, previously reported to be strong or weak biofilm producers, respectively, in mixed biofilms. Moreover, our goal was also to determine the efficacy of a cocktail of bacteriophages as biofilm control agents.Financial support is acknowledged to FCT for the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020(POCI-01-0145-FEDER-006684), RECI/BBB-EBI/0179/2012 (FCOMP-01-0124- FEDER-027462) and BioTecNorte operation (NORTE-01-0145-FEDER-000004).Catarina Milho is supported by the FCT fellowship SFRH/BD/94434/2013.info:eu-repo/semantics/publishedVersio

Antimicrobial assessment of phage therapy using a porcine model of biofilm infection

Antibiotic resistant bacterial communities persist in many types of wounds, chronic wounds in particular, in the form of biofilms. Biofilm formation is a major cause of severe infections and the main reason for a negative treatment outcome and slow healing progression. Chronic wounds are a silent epidemic essentially affecting people with co-morbid conditions such as diabetes and obesity and elderly persons particularly those with movement limitations. The development of complementary and alternative effective strategies to antibiotics for the treatment of chronic wounds is highly desired. Phage therapy constitutes a very promising approach in the control of topical microbial populations. In this work newly isolated phages were tested for their efficacy to control bacterial species that predominate in chronic wounds. Phage effectiveness was studied on 24-h old biofilms formed in polystyrene microplates and in porcine skin explants using two treatment approaches: individual phage and a cocktail of phages against four main pathogens commonly isolated from chronic wounds. The two models produced variations in the surface colonization ability, assessed by viable bacterial counts and microscopy visualization after using peptide nucleic acid (PNA) or locked nucleic acid probes (LNA) and 2-O-methyl (2-OMe) in fluorescence in situ hybridization (FISH), and in the phage-host interactions. Phages alone and combined caused greater reductions in the number of viable cells when biofilms had been formed on porcine skins and with greater variations detected at 4 h and 24 h of sampling. These results suggest that porcine skin models should be preferentially used to assess the use of phages and phage cocktails intended for topical use in order to understand the fate, throughout treatment time, of the population when dealing with biofilm-related infections.This work was supported by Portuguese Foundation for Science and Technology under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE2020(POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020–Programa Operacional Regional do Norte and the Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462). CM ac- knowledges the Portuguese Foundation for Science and Technology (FCT) grant SFRH/BD/94434/2013. SS is an Investigador FCT (IF/ 01413/2013).info:eu-repo/semantics/publishedVersio

Antimicrobials from medicinal plants: an emergent strategy to control oral biofilms

Oral microbial biofilms, directly related to oral diseases, particularly caries and periodontitis, exhibit virulence factors that include acidification of the oral microenvironment and the formation of biofilm enriched with exopolysaccharides, characteristics and common mechanisms that, ultimately, justify the increase in antibiotics resistance. In this line, the search for natural products, mainly obtained through plants, and derived compounds with bioactive potential, endorse unique biological properties in the prevention of colonization, adhesion, and growth of oral bacteria. The present review aims to provide a critical and comprehensive view of the in vitro antibiofilm activity of various medicinal plants, revealing numerous species with antimicrobial properties, among which, twenty-four with biofilm inhibition/reduction percentages greater than 95%. In particular, the essential oils of Cymbopogon citratus (DC.) Stapf and Lippia alba (Mill.) seem to be the most promising in fighting microbial biofilm in Streptococcus mutans, given their high capacity to reduce biofilm at low concentrations.The authors wish to acknowledge financial support from the project “AquaValor—Centro de Valorização e Transferência de Tecnologia da Água” (NORTE-01-0246-FEDER-000053), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF); to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020); and L. Barros thanks the national funding by FCT, P.I., through the institutional scientific employment program-contract for her contract.info:eu-repo/semantics/publishedVersio

Control of Salmonella Enteritidis on food contact surfaces with bacteriophage PVP-SE2

Salmonella is one of the worldwide leading foodborne pathogens responsible for illnesses and hospitalizations, and its capacity to form biofilms is one of its many virulence factors. This work evaluated (bacterio)phage control of adhered and biofilm cells of Salmonella Enteritidis on three different substrata at refrigerated and room temperatures, and also a preventive approach in poultry skin. PVP-SE2 phage was efficient in reducing both 24- and 48-h old Salmonella biofilms from polystyrene and stainless steel causing 2 to 5 log CFU cm2 reductions with a higher killing efficiency at room temperature. PVP-SE2 phage application on poultry skins reduced levels of Salmonella. Freezing phage-pretreated poultry skin samples had no influence on the viability of phage PVP-SE2 and their in vitro contamination with S. Enteritidis provided evidence that phages prevented their further growth. Although not all conditions favor phage treatment, this study endorses their use to prevent and control foodborne pathogen colonization of surfaces.Catarina Milho acknowledges the Portuguese Foundation for Science and Technology (FCT) grant [SFRH/BD/94434/ 2013]. Sanna Sillankorva is an Investigador FCT [IF/01413/ 2013]. This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 [POCI-01-0145-FEDER-006684] and BioTecNorte operation [NORTE-01-0145-FEDER-000004] funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte and the Project RECI/BBB-EBI/0179/2012 [FCOMP-01-0124-FEDER-027462].info:eu-repo/semantics/publishedVersio

Use of bacteriophages to prevent and control Salmonella Enteritidis biofilm formation on poultry skins at refrigerated and room temperatures

Salmonella is one of the leading worldwide foodborne pathogens responsible for illnesses and hospitalizations. Salmonellas capacity to form biofilms contributes to its resistance and persistence in both host and non-host environments, and is especially important in food processing settings. Because cross-contamination still happens during food processing and preparation, other down-stream safety measures must be applied, like the use of control agents of foodborne pathogens in food products. Phages are the natural killers of bacteria, innocuous to human and animals, and good candidates to be used in the control of bacterial pathogens. In this work we aimed to characterize a S. Enteritidis phage, phi38, which was shown to have 4.3 kbp in size, dsDNA genome and to contain 60ORFs. We also evaluated whether the addition of phi38 on poultry skin samples could decrease the levels of S. Enteritidis. For this, two approaches were used: a preventive approach focusing on decreasing Salmonella colonization ability of phage-pretreated skins; and a control one, aiming to kill Salmonella biofilms already present in the poultry skins. The effect of these two approaches was investigated at refrigerated temperatures (-18 and 4ºC) and also during 1 h at RT (22ºC). While poor effectiveness was observed using phi38 to control and reduce Salmonella biofilms following in vitro contamination of skins (< 1 log reduction of CFU) at all tested conditions, the preventive approach showed promising results (> 2 log reduction of Salmonella colonization). In this way, this study endorses that phages can be used to prevent foodborne pathogen colonization and consequently to promote food safety

Identification of type III secretion substrates of Chlamydia trachomatis using Yersinia enterocolitica as a heterologous system

BACKGROUND:Chlamydia trachomatis is an obligate intracellular human pathogen causing ocular and urogenital infections that are a significant clinical and public health concern. This bacterium uses a type III secretion (T3S) system to manipulate host cells, through the delivery of effector proteins into their cytosol, membranes, and nucleus. In this work, we aimed to find previously unidentified C. trachomatis T3S substrates.RESULTS:We first analyzed the genome of C. trachomatis L2/434 strain for genes encoding mostly uncharacterized proteins that did not appear to possess a signal of the general secretory pathway and which had not been previously experimentally shown to be T3S substrates. We selected several genes with these characteristics and analyzed T3S of the encoding proteins using Yersinia enterocolitica as a heterologous system. We identified 23 C. trachomatis proteins whose first 20 amino acids were sufficient to drive T3S of the mature form of beta-lactamase TEM-1 by Y. enterocolitica. We found that 10 of these 23 proteins were also type III secreted in their full-length versions by Y. enterocolitica, providing additional support that they are T3S substrates. Seven of these 10 likely T3S substrates of C. trachomatis were delivered by Y. enterocolitica into host cells, further suggesting that they could be effectors. Finally, real-time quantitative PCR analysis of expression of genes encoding the 10 likely T3S substrates of C. trachomatis showed that 9 of them were clearly expressed during infection of host cells.CONCLUSIONS:Using Y. enterocolitica as a heterologous system, we identified 10 likely T3S substrates of C. trachomatis (CT053, CT105, CT142, CT143, CT144, CT161, CT338, CT429, CT656, and CT849) and could detect translocation into host cells of CT053, CT105, CT142, CT143, CT161, CT338, and CT429. Therefore, we revealed several C. trachomatis proteins that could be effectors subverting host cell processes.(undefined

Antibiofilm potential of medicinal plants against Candida spp. oral biofilms: a review

The use of natural products to promote health is as old as human civilization. In recent years, the perception of natural products derived from plants as abundant sources of biologically active compounds has driven their exploitation towards the search for new chemical products that can lead to further pharmaceutical formulations. Candida fungi, being opportunistic pathogens, increase their virulence by acquiring resistance to conventional antimicrobials, triggering diseases, especially in immunosuppressed hosts. They are also pointed to as the main pathogens responsible for most fungal infections of the oral cavity. This increased resistance to conventional synthetic antimicrobials has driven the search for new molecules present in plant extracts, which have been widely explored as alternative agents in the prevention and treatment of infections. This review aims to provide a critical view and scope of the in vitro antimicrobial and antibiofilm activity of several medicinal plants, revealing species with inhibition/reduction effects on the biofilm formed by Candida spp. in the oral cavity. The most promising plant extracts in fighting oral biofilm, given their high capacity to reduce it to low concentrations were the essential oils extracted from Allium sativum L., Cinnamomum zeylanicum Blume. and Cymbopogon citratus (DC) Stapf.The authors wish to acknowledge financial support from the project “AquaValor—Centro de Valorização e Transferência de Tecnologia da Água” (NORTE-01-0246-FEDER-000053), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). The authors are also grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020); and L. Barros is grateful for her contract through the institutional scientific employment program-contract.info:eu-repo/semantics/publishedVersio

Bacteriophage IBB-PF7A loaded on sodium alginate-based films to prevent microbial meat spoilage

Despite the recent advances achieved in food industries to fulfil the growing consumer demand for high quality and food safety, microbial contamination remains a serious issue. This study aimed to incorporate IBB-PF7A bacteriophage (phage) onto sodium alginate-based films crosslinked with calcium chloride, to prevent poultry spoilage caused by Pseudomonas fluorescens. Films were prepared by casting and characterized in terms of phage loading, distribution, stability, release profile and antimicrobial performance. Results showed that phages were successfully incorporated as evidenced by their viability and homogeneous distribution within the films as assessed by microscopy. A decrease in phage viability was only detected after 8 weeks when stored under refrigerated conditions. Antimicrobial activity demonstrated that incorporated phages significantly impaired P. fluorescens growth. Films' antimicrobial efficacy was further demonstrated on chicken breast fillets artificially inoculated, decreasing 2Log P. fluorescens viable cell counts in the first two days and reductions were maintained up to 5 days of exposure (1 Log). These results highlight that phage incorporation onto sodium-alginate-based films constitutes a simple approach of preserving the antimicrobial activity of phages in a dried and insoluble format, that can further be applied in food industry for the prevention of microbial spoilage.AM, CM, MC acknowledge the Portuguese Foundation for Science and Technology (FCT) grants SFRH/BD/132911/2017, SFRH/BD/ 94434/2013, and SFRH/BD/122897/2016. SS is an Investigador FCT (IF/01413/2013). This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte and the Project RECI/BBB-EBI/0179/ 2012 (FCOMP-01-0124-FEDER-027462).info:eu-repo/semantics/publishedVersio

Electrospun nanofibres as a novel encapsulation vehicle for Felix O1 bacteriophage for new food packaging applications

Background & Objectives: Food contamination is one of the main issues in the food industry, and food packaging has been driven towards novel technologies to reduce bacterial contaminations. One of the possibilities is the development of antibacterial packaging by the encapsulation of bacteriophages, namely Felix O1, can provide protection against microbial pathogens like Salmonella Enteritidis. This work aimed the encapsulation of Felix O1 into electrospun nanofibers to be used in biodegradable packaging as a controlling agent of Salmonella Enteritidis in food products. Methods and Results: After the optimization of different electrospinning conditions (voltage, flow rate, polymer type, polymer concentration and type of solvent), polyvinyl alcohol (PVOH) with Felix O1 bacteriophage solution was electrospun on polyhydroxybutyrate/polyhydroxyvalerate film forming a layer composed by sub-micro nanofibers. The optimized conditions were: SM buffer solution of PVOH at 14% (w/v) at a flow rate 0.3 mL/h and applied voltage of 25 kV. Conclusions: After the formation of nanofibers with a size around 100 nm (observed through Scanning Electron Microscopy), release tests in SM buffer for 1 h revealed a high bacteriophage viability (105), but still, there was a decrease of one log in phage titre. DSC and TGA results revealed differences between the films and the films with nanofibers, showing the influence of the nanofibers in the system namely in the thermal behavior. Results show that this new packaging system is promising for the development of active packaging using bacteriophages. Significance and impact of the study: The encapsulation of bacteriophages through electrospinning shows high potential to be explored as a new feature in food packaging, as a possible solution for bacterial contamination in foods.info:eu-repo/semantics/publishedVersio