Browsing by Author "Sousa, Mariana"
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- Assessing the persister state in Listeria monocytogenes in response to gentamicin, benzalkonium chloride and bacteriocin SB83Publication . Sousa, Mariana; Azevedo, Mónica; Magalhães, Rui; Almeida, Gonçalo; Teixeira, PaulaIntroduction: In the context of food safety, persister cells are a public health concern since these may contribute to the persistence and survival of bacteria in food processing environments. Listeria monocytogenes persisters are described as a subpopulation of growth-arrested cells that possess non-heritable, temporary tolerance to lethal doses of antibiotics (e.g., gentamicin) and can revert to an active state under favorable conditions. This dormancy state can be studied through biphasic killing curves – initial drop of susceptible cells followed by a plateau of persisters in response to continuous antibiotic exposure or to its increasing concentration. Nonewithstanding, gaps still exist to understand whether stresses present in the food-producing sector, such as disinfectants and bacteriocins, can also contribute to the formation of persisters. Conclusion: Neither bacteriocin SB83 nor BAC were able to induce the persisters state.The gentamicin exposure is still the only validated approach to achieve the persister dormancy state. Further studies must focus in understanding the tolerance profile of persister cells to food facilities stresses.
- Assessing the persister state to benzalkonium chloride in Listeria monocytogenesPublication . Sousa, Mariana; Azevedo, Mónica; Magalhães, Rui; Almeida, Gonçalo; Teixeira, PaulaIntroduction: Listeria monocytogenes (Lm) persisters are a subpopulation of growth-arrested cells that possess non-heritable, temporary tolerance to lethal doses of antibiotics (e.g., gentamicin), which can be studied through biphasic killing curves – initial drop of susceptible cells followed by a plateau of persisters in response to continuous antibiotic exposure or to its increasing concentration. In the context of food safety, persister cells may contribute to the persistence and survival of bacteria in relevant environments, such as in the food-producing sector. Few studies have been published on Lm persisters and most only address antibiotic exposure, while information on other stresses that cells are exposed to in these environments (e.g. disinfectants, NaCl concentrations, pH) is lacking. Preliminary objective: Can persister cells be formed in response to benzalkonium chloride (BAC)? Results: The dose-dependent killing curves for BAC of four Lm strains isolated from a cheese factory, with known minimum inhibitory concentrations (1.25 mg/L), are represented in Figure 1. All strains show an initial plateau in CFU counts up to 12.5 mg/L, followed by a subsequent decrease and eventual death. This does not correspond to the expected persister curves (described in introduction). However, it is worth noting that these strains were able to survive in BAC concentrations 10-fold higher than the MIC, which may indicate (i) a tolerance equivalent to that of persister cells; (ii) that these concentrations are below the minimum bactericidal concentration; or (iii) that instead of entering a state of persisters, cells enter a viable but non-culturable state. Conclusion: BAC by itself does not induce the persister state of these Lm strains. Additional studies are being conducted with different stresses encountered in food processing environments.
- Assessing the virulence potential of different Listeria monocytogenes Clonal Complexes with Galleria mellonella larvae modelPublication . Sousa, Mariana; Silva, Sónia; Araújo, Daniela; Castro, Joana; Almeida, Gonçalo; Ventura, Catarina; Magalhães, Rui; Ferreira, Vânia; Teixeira, PaulaIntroduction and Objective: Listeria monocytogenes is a pathogen causes infection mainly through food contamination. This species is well-known for its diversity, exhibiting differential virulence potential within strains from different clonal complexes (CCs). These can be defined as hypervirulent CCs, that are highly frequent among clinical cases and cause severe clinical outcomes (i.e. maternal-neonatal and central nervous system infections), or as hypovirulent CCs, that are associated with food and food processing environments and present attenuated virulence, causing disease in highly immunocompromised individuals. Many methodologies have been employed to study this differentiated pathogenicity, with Galleria mellonella larvae emerging as an in vivo infection model [1, 2]. The purpose of this study was to assess the pathogenic potential of L. monocytogenes isolates from eight distinct CCs, either hyper- and hypovirulent, through an in vivo infection and further analysis of its impact on the immune response of Galleria larvae. Methodology: Sixteen L. monocytogenes isolates from hypervirulent (CC1, CC2, CC4, CC6, CC87 and CC388) or hypovirulent (CC9 and CC121) CCs were selected for the purposes of this study. An infectious dose of 1x107 CFU/mL of L. monocytogenes was injected in the hindmost left proleg, into the hemocoel of the larvae. Results: The survival curves and the health index scores of the G. mellonella larvae infected with the 16 selected L. monocytogenes isolates are represented, respectively, in Figure 1A and 1B. The gathered results showed that: The two L. monocytogenes isolates that caused the highest and lowest survival rates are from hypervirulent CCs; Significant differences intra-clonal complex were observed within the CC6; Isolates from CC9 exhibited a hypervirulent phenotype when injected into G. mellonella larvae; At all time points, a decrease in the HISs was observed for larvae injected with each isolate. The observed HIS pattern was similar to that noted on the survival curves. Considering the pathogenic capacity of L. monocytogenes strains, a subset of five isolates was selected to pursue with further analysis of the host’s immune response based on the expression of the genes coding for antimicrobial peptides (AMPs). The obtained results showed an AMPs overexpression in strains that caused lower survival rates. Conclusions: The results showed that no correlation between hyper- or hypovirulent CCs and survival rates or HIS, as isolates causing higher and lower survival were from hypervirulent CCs. Additionally, the virulence potential of L. monocytogenes strains seems to be strain-dependent upon infection of G. mellonella larvae. Further studies need to be conducted with a larger sample size to draw more robust conclusions. Additionally, the expression of AMPs should be monitored throughout the infection process along with bacterial cells levels.
- Current methodologies available to evaluate the virulence potential among Listeria monocytogenes clonal complexesPublication . Sousa, Mariana; Magalhães, Rui; Ferreira, Vânia; Teixeira, PaulaListeria monocytogenes is a foodborne pathogen that causes listeriosis in humans, the severity of which depends on multiple factors, including intrinsic characteristics of the affected individuals and the pathogen itself. Additionally, emerging evidence suggests that epigenetic modifications may also modulate host susceptibility to infection. Therefore, different clinical outcomes can be expected, ranging from self-limiting gastroenteritis to severe central nervous system and maternal-neonatal infections, and bacteremia. Furthermore, L. monocytogenes is a genetically and phenotypically diverse species, resulting in a large variation in virulence potential between strains. Multilocus sequence typing (MLST) has been widely used to categorize the clonal structure of bacterial species and to define clonal complexes (CCs) of genetically related isolates. The combination of MLST and epidemiological data allows to distinguish hypervirulent CCs, which are notably more prevalent in clinical cases and typically associated with severe forms of the disease. Conversely, other CCs, termed hypovirulent, are predominantly isolated from food and food processing environments and are associated with the occurrence of listeriosis in immunosuppressed individuals. Reports of genetic traits associated with this diversity have been described. The Food and Agriculture Organization (FAO) is encouraging the search for virulence biomarkers to rapidly identify the main strains of concern to reduce food waste and economical losses. The aim of this review is to comprehensively collect, describe and discuss the methodologies used to discriminate the virulence potential of L. monocytogenes CCs. From the exploration of in vitro and in vivo models to the study of expression of virulence genes, each approach is critically explored to better understand its applicability and efficiency in distinguishing the virulence potential of the pathogen.
- High salinity and temperature contribute to Listeria monocytogenes persistence by inducing the VBNC statePublication . Azevedo, Mónica; Sousa, Mariana; Magalhães, Rui; Almeida, Gonçalo; Teixeira, PaulaIntroduction: Foodborne listeriosis has a low case rate but high mortality, posing a public health concern. Listeria monocytogenes can contaminate food-processing and storage environments, compromising the food supply chain. L. monocytogenes adapts to stresses like nutrient deprivation, extreme temperatures, and high salinity. Understanding its persistence mechanisms, including the ability to enter a viable but non-culturable (VBNC) state, is essential. Discussion: Microscopy using the BacLight assay showed significantly higher counts of viable cells compared to culturable cells detected using TSA and PALCAM, indicating the presence of VBNC cells. At 37 ºC, no colonies were observed with the plating method, yet viable cell counts remained high (BacLight assay). Similarly, at 8 ºC, viable cells outnumbered culturable bacteria. However, the low metabolic activity of L. monocytogenes at this temperature makes it less likely to enter the VBNC state. These results highlight the environmental conditions that promote the VBNC state in L. monocytogenes, offering insights for improving long-term storage, sterilization, and control measures in food products. Additionally, the findings emphasize the limitations of current culture-based methods in detecting VBNC bacteria, underscoring the need for more advanced detection techniques. Conclusion: Prolonged exposure to nutrient deficiencies, and high NaCl concentrations and temperature can induce the VBNC state of L. monocytogenes. VBNC cells evade detection by standard growth- based monitoring methods, thus improved detection methods, such as molecular screening techniques, are needed.
- In the quest for reliable tools to explore the virulence potential of Listeria monocytogenes clonal complexesPublication . Sousa, Mariana; Magalhães, Rui; Ferreira, Vânia; Teixeira, PaulaIntroduction and objective: Listeria monocytogenes is a foodborne pathogen that causes human listeriosis. This pathogen is characterized by an intra-species heterogeneity so its strains can be grouped into clonal complexes (CCs) that are defined either as hypervirulent – mainly associated with clinical cases or hypovirulent – associated with food or food processing environments and with infections in susceptible groups [1]. The impact of L. monocytogenes extends beyond health concerns to economic implications since detection of contaminated food results in production interruptions and product recalls. Considering the persistence of L. monocytogenes in the food industry, there is an urgent need to define reliable biomarkers and to determine whether CC characterization is robust enough to discriminate high-risk strains. Listeria monocytogenes has a typical β-hemolytic profile, which is outlined by crucial virulence factors (hly and prfA genes), however, some strains exhibiting non- hemolytic or attenuated hemolysis have been reported [2]. The aim of this work was to investigate if these phenotypes could be associated with hypovirulent CCs.
- Papel profissional enquanto diagnóstico de Enfermagem no contexto escolar – Um estudo de casoPublication . Silva, Andreia; Sousa, Mariana; Melo, Pedro; Cardoso, Teresa; Martins, Alice; Sousa, SérgioA escola é um setting de excelência na promoção de comportamentos saudáveis ao longo da vida. Os encarregados de educação e os docentes desempenham um papel fundamental na proteção da saúde e no desenvolvimento dos adolescentes no âmbito das decisões e comportamentos. No âmbito da elaboração de um projeto de intervenção em contexto escolar, perspetiva-se uma abordagem longitudinal (2014-2023) fundamentada em três domínios: comportamentos associados ao consumo de substâncias psicoativas; comportamentos aditivos sem substância; comportamentos associados à sexualidade. Na primeira etapa de planeamento em saúde, no diagnóstico de situação, definiu-se a tomada de decisão clínica em enfermagem em três focos de atenção: o Processo de Tomada de Decisão nos alunos; o Papel Profissional nos docentes e o Papel Parental nos encarregados de educação. Neste artigo descrevemos o Papel Profissional dos docentes, enquanto diagnóstico de enfermagem, no âmbito dos domínios em estudo, utilizando como método o estudo de caso
- Understanding virulence variability among Listeria monocytogenes Clonal ComplexesPublication . Sousa, Mariana; Magalhães, Rui; Ferreira, Vânia; Teixeira, PaulaIntroduction and objective: Listeria monocytogenes is a foodborne pathogen that causes human listeriosis. This pathogen is characterized by an intra-species heterogeneity so, its strains can be grouped into clonal complexes (CCs) defined either as hypervirulent – mainly associated with clinical cases, or hypovirulent – associated with food or food processing environments and causing infections mainly in highly susceptible individuals. This pathogen has a complex infection mechanism represented in Figure 1. A crucial step for infection is the internalization of the pathogen into host cells, which is accomplished by the linkage of internalins (A and B) to host receptors. The differences in virulence potential may be explained by the presence of mutations in key virulence genes. The purpose of this study was to explore the virulence capacity based of L. monocytogenes CCs through in vitro infection assays and to further assess mutations in the inlA gene, which is crucial for the invasion into intestinal epithelial cells by the pathogen. Methodology: Eleven isolates were selected based on the top five hypervirulent CCs occurring in Portugal and one strain from CC4 was also included, as this hypervirulent CC is one of the best characterized CCs around Europe. Five isolates from hypovirulent CCs were also used. Invasion assays were performed in Caco-2 cells and subsequently the presence of PMSC mutations in the inlA gene were assessed, using the MEGA software (version 10.1.8). Results: Our results show a clear-cut difference on invasion capacity between isolates from hyper- and hypovirulent CCs, with the latter showing significantly reduced efficiencies. Sequence analysis of the inlA gene, revealed that only hypovirulent CCs carried PMSC mutations, leading to truncated forms of the InlA protein. A 3-codon deletion was detected in isolates from CC6.