This study provides a more accurate comparison between the antibiotic susceptibilities of planktonic versus biofilm populations, because the cell densities in the two populations were similar and because we measured the concentration required to inhibit bacterial metabolism rather than to eradicate the entire bacterial population. While the biofilm phenotype is highly resistant to antibiotics that target cell wall synthesis, it is fairly susceptible to antibiotics that target RNA and protein synthesis.

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.

Candida albicans secreted aspartyl proteinases (Saps) are considered virulence-associated factors. Several members of the Sap family were claimed to play a significant role in the progression of candidiasis established by the hematogenous route. This assumption was based on the observed attenuated virulence of sap-null mutant strains. However, the exclusive contribution of SAP genes to their attenuated phenotype was not unequivocally confirmed, as the Ura status of these mutant strains could also have contributed to the attenuation. In this study, we have reassessed the importance of SAP1 to SAP6 in a murine model of hematogenously disseminated candidiasis using sap-null mutant strains not affected in their URA3 gene expression and compared their virulence phenotypes with those of Ura-blaster sap mutants. The median survival time of BALB/c mice intravenously infected with a mutant strain lacking SAP1 to SAP3 was equivalent to that of mice infected with wild-type strain SC5314, while those infected with mutant strains lacking SAP5 showed slightly extended survival times. Nevertheless, no differences could be observed between the wild type and a ⌬sap456 mutant in their abilities to invade mouse kidneys. Likewise, a deficiency in SAP4 to SAP6 had no noticeable impact on the immune response elicited in the spleens and kidneys of C. albicans-infected mice. These results contrast with the behavior of equivalent Ura-blaster mutants, which presented a significant reduction in virulence. Our results suggest that Sap1 to Sap6 do not play a significant role in C. albicans virulence in a murine model of hematogenously disseminated candidiasis and that, in this model, Sap1 to Sap3 are not necessary for successful C. albicans infection.The polymorphic yeast Candida albicans is an important opportunistic human pathogen causing infections that range from superficial mucosal lesions to life-threatening systemic disease. It is by far the most common cause of fungal invasive infections, which could be attributed to the little immunosuppression required to predispose an individual to invasive Candida infections (39). Host physical barriers and immune system integrity are crucial factors in controlling the establishment of infection. However, the high adaptability of C. albicans to different host niches, by the expression of appropriate sets of virulence-related genes, is also a determinant (19, 51). Several of these virulence attributes may participate in and influence the infective process, depending on the site and stage of invasion and on the nature of the host response (37). The secretion of hydrolytic enzymes during infection is required as a virulence attribute to aid adhesion, invasion, and the destruction of host immune factors, in addition to nutrient acquisition (21).Among these enzymes, secreted aspartyl proteinases (Sap), encoded by a 10-member gene family (SAP1 to SAP10) have been the most extensively studied (35). The 10 SAP genes that compose this family can be divided into subfamilies based on amino acid sequence ho...

Staphylococcus epidermidis biofilms with different proportions of viable but nonculturable bacteria were used to show that SYBR green (SYBR) may be used as a probe to evaluate the bacterial physiological state using flow cytometry. Biofilms grown in excess glucose presented significantly higher proportions of dormant bacteria than biofilms grown in excess glucose with buffered pH conditions or with exponential-phase planktonic cultures. Bacteria obtained from biofilms with high or low proportions of viable but nonculturable cells were further cultured in broth medium and stained with SYBR at different time points. An association between bacterial growth and SYBR staining intensity was observed. In addition, bacteria presenting higher SYBR fluorescence intensity also stained more intensely with cyanoditolyl tetrazolium chloride, used as a probe to evaluate cellular metabolism. Accordingly, planktonic bacteria treated with rifampicin, an inhibitor of bacterial RNA transcription, presented lower SYBR and cyanoditolyl tetrazolium chloride staining intensity than nontreated bacteria. Overall, our results indicate that SYBR, in addition to being used as a component of LIVE/DEAD stain, may also be used as a probe to evaluate the physiological state of S. epidermidis cells.

The hallmark of familial amyloid polyneuropathy (FAP) is the presence of extracellular deposits of transthyretin (TTR) aggregates and amyloid fibers in several tissues, particularly in the peripheral nervous system. The molecular pathways to neurodegeneration in FAP still remain elusive; activation of nuclear factor B, pro-inflammatory cytokines, oxidative stress, and pro-apoptotic caspase-3 has been demonstrated "in vivo" in clinical samples and in cell culture systems. In this study, we investigated the involvement of endoplasmic reticulum (ER) stress response in FAP by showing activation of the classical unfolded protein response pathways in tissues not specialized in TTR synthesis but presenting extracellular TTR aggregate and fibril deposition. We also proved cytotoxicity by Ca 2؉ efflux from the ER in cell cultures incubated with TTR oligomers. Taken together, these studies evidence ER stress associated with a extracellular signal in a misfolding disorder.The hallmark of familial amyloid polyneuropathy (FAP) 2 is the extracellular deposition of TTR in several tissues, particularly in the peripheral nervous system (1). The most common FAP-related point mutation in TTR is the substitution of a valine for a methionine at position 30 of the polypeptide chain (2). It was previously described that non-fibrillar TTR aggregates were present in sciatic nerves of asymptomatic individuals, which also presented increased caspase-3 activation (3). Other stress-related molecules were also found to be linked to TTR aggregate-induced degeneration, namely pro-inflammatory cytokines and the nuclear factor B (NF-B) (4, 5).Cellular stress conditions including altered redox status, increased protein synthesis, expression of misfolded proteins, and perturbation of calcium homeostasis can induce endoplasmic reticulum (ER)-specific stress response (6). This adaptive response is known as unfolded protein response and includes three basic signal transduction pathways mediated by the inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6), and double-stranded RNA-activated protein kinaselike ER kinase (PERK). All the main transducers are activated in response to accumulation of unfolded proteins, in a process dependent on the ER-resident chaperone BiP. BiP is a member of the heat-shock protein 70 family that transiently binds to newly synthesized, misfolded, or unassembled proteins in the ER lumen (7). The more effective chemical inducers of BiP expression are tunicamycin (which blocks N-glycosylation), thapsigargin (inhibitor of the ER Ca 2ϩ ATPase), and calcium ionophores, all affecting the ER folding capacity.It is now widely accepted that the specific induction of BiP is indicative of ER stress due to its key role in the regulation of ER stress signaling. In non-stressed conditions, BiP binds to the luminal domains of IRE1, PERK, and ATF6, keeping them in the ER. Upon ER stress, BiP is released from IRE1 and PERK, permitting their oligomerization, phosphorylation, and activation (8 -10), and ATF6 is transp...

The proportion of dormant bacteria within Staphylococcus epidermidis biofilms may determine its inflammatory profile. Previously, we have shown that S. epidermidis biofilms with higher proportions of dormant bacteria have reduced activation of murine macrophages. RNA-sequencing was used to identify the major transcriptomic differences between S. epidermidis biofilms with different proportions of dormant bacteria. To accomplish this goal, we used an in vitro model where magnesium allowed modulation of the proportion of dormant bacteria within S. epidermidis biofilms. Significant differences were found in the expression of 147 genes. A detailed analysis of the results was performed based on direct and functional gene interactions. Biological processes among the differentially expressed genes were mainly related to oxidation-reduction processes and acetyl-CoA metabolic processes. Gene set enrichment revealed that the translation process is related to the proportion of dormant bacteria. Transcription of mRNAs involved in oxidation-reduction processes was associated with higher proportions of dormant bacteria within S. epidermidis biofilm. Moreover, the pH of the culture medium did not change after the addition of magnesium, and genes related to magnesium transport did not seem to impact entrance of bacterial cells into dormancy.

Neospora caninum is a coccidian parasite causative of clinical infections in a wide range of animal hosts. The maturation and activation of splenic conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) were studied here in BALB/c mice challenged intraperitoneal with N. caninum tachyzoites. The number of cDCs was found to decrease in the spleen of the infected mice 12 h and 2 days after the parasitic challenge, whereas at day 5 after infection it was significantly above that of mock-infected controls. In contrast, the number of splenic pDCs did not change significantly on infection. In the infected mice, both cell subtypes displayed an activated phenotype with upregulation of costimulatory and MHC class II molecules. This stimulatory effect was more marked at the earliest assessed time point after infection, 12 h, when a clear increase in the frequency of cDCs (CD8a + and CD8a À ) and pDCs producing interleukin-12 (IL-12) was also observed. N. caninum tachyzoites could be observed by confocal microscopy associated with sorted DCs. Overall, these results present the first evidence that both cDCs and pDCs mediate in vivo the innate immune response to N. caninum infection through the production of IL-12, a key cytokine for host resistance to neosporosis.

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. use of medical resources and, consequently, increased healthcare costs (Dimick et al., 2001; Rogers et al., 2009). Critically ill immune-compromised patients (Bearman & Wenzel, 2005) and premature neonates (Fallat et al., 1998) are the individuals most vulnerable to this opportunistic pathogen. S. epidermidis biofilm formation involves initial cellular adherence to a surface followed by intercellular aggregation and accumulation in multilayer cell clusters (Otto, 2009). This process is dependent on the synthesis of adhesive extracellular molecules (Götz, 2002), such as the polysaccharide intercellular adhesin (PIA), also known as poly-Nacetylglucosamine (PNAG), a major constituent mediating cell-to-cell adhesion in staphylococci (Mack et al., 1994, Abbreviations: BMDC, bone marrow-derived dendritic cell; MFI, mean fluorescence intensity; PI, propidium iodide; qPCR, quantitative PCR.