Ângela Maria Oliveira Sousa França

Using an in-vitro biofilm model to assess the virulence potential of Bacterial Vaginosis or non-Bacterial Vaginosis Gardnerella vaginalis isolates

Gardnerella vaginalis is the most common species found in bacterial vaginosis (BV). However, it is also present in a significant proportion of healthy women and G. vaginalis vaginal colonization does not always lead to BV. In an effort to better understand the differences between G. vaginalis isolated from women with a positive (BV) versus a negative (non-BV) diagnosis of BV, we compared the virulence potential of 7 BV and 7 non-BV G. vaginalis isolates and assessed the virulence factors related to biofilm formation, namely: initial adhesion and cytotoxic effect, biofilm accumulation, susceptibility to antibiotics, and transcript levels of the known vaginolysin, and sialidase genes. Furthermore, we also determined the ability of G. vaginalis to displace lactobacilli previously adhered to HeLa cells. Our results showed that non-BV strains were less virulent than BV strains, as suggested by the lower cytotoxicity and initial adhesion to Hela cells. Significant differences in expression of known virulence genes were also detected, further suggesting a higher virulence potential of the BV associated G. vaginalis. Importantly, we demonstrated that BV associated G. vaginalis were able to displace pre-coated vaginal protective lactobacilli and we hypothesize this to be a trigger for BV development.

Dormant bacteria within Staphylococcus epidermidis biofilms have low inflammatory properties and maintain tolerance to vancomycin and penicillin after entering planktonic growth

Staphylococcus epidermidis is the most commonly isolated aetiological agent of nosocomial infections, mainly due to its ability to establish biofilms on indwelling medical devices. Detachment of bacteria from S. epidermidis biofilms and subsequent growth in the planktonic form is a hallmark of the pathogenesis of these infections leading to dissemination. Here we showed that S. epidermidis cells collected from biofilms cultured in conditions that promote cell viability present marked changes in their physiological status upon initiating a planktonic mode of growth. When compared to cells growing in biofilms, they displayed an increased SYBR green I staining intensity, increased transcription of the rpiA gene, decreased transcription of the icaA gene, as well as higher susceptibility to vancomycin and penicillin. When bacteria collected from biofilms with high proportions of dormant cells were subsequently cultured in the planktonic mode, a large proportion of cells maintained a low SYBR green I staining intensity and increased resistance to vancomycin and penicillin, a profile typical of dormant cells. This phenotype further associated with a decreased ability of these biofilm-derived cells to induce the production of pro-inflammatory cytokines by bone marrow-derived dendritic cells in vitro. These results demonstrated that cells detached from the biofilm maintain a dormant cell-like phenotype, having a low pro-inflammatory effect and decreased susceptibility to antibiotics, suggesting these cells may contribute to the recalcitrant nature of biofilm infections.

Evidence for inter- and intraspecies biofilm formation variability among a small group of coagulase-negative staphylococci

Coagulase-negative staphylococci (CoNS) are common bacterial colonizers of the human skin. They are often involved in nosocomial infections due to biofilm formation in indwelling medical devices. While biofilm formation has been extensively studied in Staphylococcus epidermidis, little is known regarding other CoNS species. Here, biofilms from six different CoNS species were characterized in terms of biofilm composition and architecture. Interestingly, the ability to form a thick biofilm was not associated with any particular species, and high variability on biofilm accumulation was found within the same species. Cell viability assays also revealed different proportions of live and dead cells within biofilms formed by different species, although this parameter was particularly similar at the intraspecies level. On the other hand, biofilm disruption assays demonstrated important inter- and intraspecies differences regarding extracellular matrix composition. Lastly, confocal laser scanning microscopy experiments confirmed this variability, highlighting important differences and common features of CoNS biofilms. We hypothesized that the biofilm formation heterogeneity observed was rather associated with biofilm matrix composition than with cells themselves. Additionally, our results indicate that polysaccharides, DNA and proteins are fundamental pieces in the process of CoNS biofilm formation.

Modulation of poly-N-acetylglucosamine accumulation within mature Staphylococcus epidermidis biofilms grown in excess glucose

PNAG is a major component of Staphylococcus epidermidis biofilms involved in intercellular adhesion as well as in the interaction of the biofilm with components of the host immune response. Synthesis of PNAG has been found to be regulated by several environmental factors. In the present study, the effect of glucose metabolism‐dependent culture medium acidification in PNAG accumulation was evaluated. Established S. epidermidis biofilms were allowed to grow in excess glucose with or without maintained pH conditions. PNAG accumulation in these biofilms was determined by flow cytometry and fluorescence microscopy using wheat germ agglutinin as a fluorescent probe. Biofilms grown in maintained pH conditions presented significantly higher amounts of this polymer as well as higher icaA expression than biofilms grown in acidic pH conditions. Moreover, PNAG accumulation in biofilms grown in non‐maintained pH conditions occurred in association with cell death. Overall, we show that glucose metabolism by decreasing the culture pH affects biofilm physiology in respect to PNAG production and cell death. The reported in vitro modulation of PNAG accumulation within S. epidermidis biofilms further highlights the role of environment on determining the biofilm physiological state.

Staphylococcus epidermidis is largely dependent on iron availability to form biofilms

Staphylococcus epidermidis has long been known as a major bacterial coloniser of the human skin, yet it is also a prominent nosocomial pathogen. Its remarkable ability to assemble structured biofilms has been its major known pathogenic feature to date. Notwithstanding important discoveries that have been accomplished, several questions about S. epidermidis biofilm formation still remain to be elucidated. This study aimed to assess whether iron availability modulates S. epidermidis biofilm formation and, if so, to explore how such modulation occurs. Biofilms of three S. epidermidis strains were grown under iron-enriched/-deficient conditions and several physiologic and transcriptomic changes were assessed. Our data revealed that while physiologic iron levels do not compromise biofilm formation, iron excess or deficiency is detrimental for this process. Conversely, biofilm cells were not affected in the same way when grown planktonically. By studying biofilm cells in detail we found that their viability and cultivability were seriously compromised by iron deficiency. Also, a temporal analysis of biofilm formation revealed that iron excess/deficiency: i) impaired biomass accumulation from 6h onwards, and ii) induced changes in the biofilm structure, indicating that iron availability plays a pivotal role from an early biofilm development stage. The expression of several putative iron-related genes, namely encoding siderophore biosynthesis/transport-related proteins, was found to be modulated by iron availability, providing a biological validation of their function on S. epidermidis iron metabolism. This study therefore provides evidence that iron plays a pivotal role on S. epidermidis biofilm formation.