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.

Staphylococcus epidermidis is an opportunistic pathogen and, due to its ability to establish biofilms, is a leading causative agent of indwelling medical device-associated infection. The presence of high amounts of dormant bacteria is a hallmark of biofilms, making them more tolerant to antimicrobials and to the host immune response. We observed that S. epidermidis biofilms grown in excess glucose accumulated high amounts of viable but non-culturable (VBNC) bacteria, as assessed by their low ratio of culturable bacteria over the number of viable bacteria. This effect, which was a consequence of the accumulation of acidic compounds due to glucose metabolism, was counteracted by high extracellular levels of calcium and magnesium added to the culture medium allowing modulation of the proportions of VBNC bacteria within S. epidermidis biofilms. Using bacterial inocula obtained from biofilms with high and low proportions of VBNC bacteria, their stimulatory effect on murine macrophages was evaluated in vitro and in vivo. The inoculum enriched in VBNC bacteria induced in vitro a lower production of tumour necrosis factor alpha, interleukin-1 and interleukin-6 by bone-marrow-derived murine macrophages and, in vivo, a lower stimulatory effect on peritoneal macrophages, assessed by increased surface expression of Gr1 and major histocompatibility complex class II molecules. Overall, these results show that environmental conditions, such as pH and extracellular levels of calcium and magnesium, can induce dormancy in S. epidermidis biofilms. Moreover, they show that bacterial suspensions enriched in dormant cells are less inflammatory, suggesting that dormancy can contribute to the immune evasion of biofilms.

Aims While numerous studies have reported on nanoparticle uptake by phagocytic cells, the mechanisms of this uptake are poorly understood. A metastudy of research focusing on biological particulate matter has postulated that nanoparticles cannot be phagocytosed and therefore must enter cells via pinocytosis. The purpose of this study was to identify the route(s) of uptake of gold nanoparticles in vitro and to determine if these route(s) depend on particle size. Materials & Methods The parent RAW264.7 cell line and its derivatives, transduced with a virus carrying siRNA to macrophage scavenger receptor A, were used as model phagocytes. Citrate-stabilized gold colloids were used as model nanoparticles. We used chemical inhibitors known to interfere with specific routes of particulate uptake. We developed multifocal light microscopy methods including multifocal stack analysis with NIH ImageJ software to analyze cell uptake. Results Irrespective of size, gold nanoparticles are internalized by macrophages via multiple routes, including both phagocytosis and pinocytosis. If either route was blocked, the particles entered cells via the other route. Conclusion Gold nanoparticles with hydrodynamic sizes below 100 nm can be phagocytosed. Phagocytosis of anionic gold colloids by RAW264.7 cells is mediated by macrophage scavenger receptor A.

Nanoparticles (NPs) can offer many advantages over traditional drug design and delivery, as well as toward medical diagnostics. As with any medical device or pharmaceutical drug intended to be used for in vivo biomedical applications, NPs must be sterile. However, very little is known regarding the effect of sterilization methods on the intrinsic properties and stability of NPs. Herein a detailed analysis of physicochemical properties of two types of AuNPs upon sterilization by means of five different techniques is reported. In addition, cell viability and production of reactive oxygen species are studied. The results indicate that sterilization by ethylene oxide seems to be the most appropriate technique for both types of NPs. It is concluded that it is crucial to test several methods in order to establish the specific type of sterilization to be performed for each particular NP.

Bacterial vaginosis is the most common gynecological disorder affecting women of reproductive age. Bacterial vaginosis is frequently associated with the development of a Gardnerella vaginalis biofilm. Recent data indicates that G. vaginalis biofilms are more tolerant to antibiotics and are able to incorporate other bacterial vaginosis -associated species, yielding a multi-species biofilm. However, despite its apparent role in bacterial vaginosis, little is known regarding the molecular determinants involved in biofilm formation by G. vaginalis. To gain insight into the role of G. vaginalis in the pathogenesis of bacterial vaginosis, we carried out comparative transcriptomic analysis between planktonic and biofilm phenotypes, using RNA-sequencing. Significant differences were found in the expression levels of 815 genes. A detailed analysis of the results obtained was performed based on direct and functional gene interactions. Similar to other bacterial species, expression of genes involved in antimicrobial resistance were elevated in biofilm cells. In addition, our data indicate that G. vaginalis biofilms assume a characteristic response to stress and starvation conditions. The abundance of transcripts encoding proteins involved in glucose and carbon metabolism was reduced in biofilms. Surprisingly, transcript levels of vaginolysin were reduced in biofilms relative to planktonic cultures. Overall, our data revealed that gene-regulated processes in G. vaginalis biofilms resulted in a protected form of bacterial growth, characterized by low metabolic activity. This phenotype may contribute towards the chronic and recurrent nature of bacterial vaginosis. This suggests that G. vaginalis is capable of drastically adjusting its phenotype through an extensive change of gene expression.

Both dynamic and fed-batch systems have been used for the study of biofilms. Dynamic systems, whose hallmark is the presence of continuous flow, have been considered the most appropriate for the study of the last stage of the biofilm lifecycle: biofilm disassembly. However, fed-batch is still the most used system in the biofilm research field. Hence, we have used a fed-batch system to collect cells released from Staphylococcus epidermidis biofilms, one of the most important etiological agents of medical device-associated biofilm infections. Herein, we showed that using this model it was possible to collect cells released from biofilms formed by 12 different S. epidermidis clinical and commensal isolates. In addition, our data indicated that biofilm disassembly occurred by both passive and active mechanisms, although the last occurred to a lesser extent. Moreover, it was observed that S. epidermidis biofilm-released cells presented higher tolerance to vancomycin and tetracycline, as well as a particular gene expression phenotype when compared with either biofilm or planktonic cells. Using this model, biofilm-released cells phenotype and their interaction with the host immune system could be studied in more detail, which could help providing significant insights into the pathophysiology of biofilm-related infections.

Staphylococcus epidermidis biofilm-related infections are a current concern within the medical community due to their high incidence and prevalence, particularly in patients with indwelling medical devices. Biofilm gene expression analysis by quantitative real-time PCR (qPCR) has been increasingly used to understand the role of biofilm formation in the pathogenesis of S. epidermidis infections. However, depending on the RNA extraction procedure, and cDNA synthesis and qPCR master mixes used, gene expression quantification can be suboptimal. We recently showed that some RNA extraction kits are not suitable for S. epidermidis biofilms, due to sample composition, in particular the presence of the extracellular matrix. In this work, we describe a custom RNA extraction assay followed by the evaluation of gene expression using different commercial reverse transcriptase kits and qPCR master mixes. Our custom RNA extraction assay was able to produce good quality RNA with reproducible gene expression quantification, reducing the time and the costs associated. We also tested the effect of reducing cDNA and qPCR reaction volumes and, in most of the cases tested, no significant differences were found. Finally, we titered the SYBR Green I concentrations in standard PCR master mixes and compared the normalized expression of the genes icaA, bhp, aap, psmβ1 and agrB using 4 distinct biofilm forming S. epidermidis strains to the results obtained with commercially available kits. The overall results demonstrated that despite some statistically, but not biologically significant differences observed, the customized qPCR protocol resulted in the same gene expression trend presented by the commercially available kits used.

Coagulase-negative staphylococci (CoNS) have emerged as major pathogens in healthcare-associated facilities, being S. epidermidis, S. haemolyticus and, more recently, S. lugdunensis, the most clinically relevant species. Despite being less virulent than the well-studied pathogen S. aureus, the number of CoNS strains sequenced is constantly increasing and, with that, the number of virulence factors identified in those strains. In this regard, biofilm formation is considered the most important. Besides virulence factors, the presence of several antibiotic-resistance genes identified in CoNS is worrisome and makes treatment very challenging. In this review, we analyzed the different aspects involved in CoNS virulence and their impact on health and food.