Detection of Epstein-Barr virus infection in primary junctional epithelial cell cultures.

Background: Junctional epithelium (JE) provides the front-line defense against pathogens invading periodontium. The breakdown of the JE barrier is the hallmark of periodontitis. Recent studies have implicated the Epstein-Barr virus (EBV) as one of the etiopathogenetic factors of periodontitis. EBV exhibits tropism for two target cells in vivo: B cells, where it primarily remains latent, and epithelial cells, where viral replication occurs. Objective: Our knowledge of junctional epithelial cell (JEC) infection with EBV has been limited by the difficulty of generating cell cultures and the inability to infect JECs in vitro readily. Design: To study EBV infection in JECs, we developed human JEC cultures derived from a periodontitis patient. Furthermore, we established a successful contact-free co-culture infection model between the EBV-donor B95-8 cell line and the EBV-permissive JEC culture. JECs and EBV infection of JECs were detected using immunofluorescent staining of cytokeratin 19 and EBNA1, respectively. In addition, EBV infection was confirmed by RT-qPCR for EBNA1, LMP1, and BZLF1 expression. Results and conclusions: Our results suggest that the infection of JECs with EBV can occur in an in vitro experimental model. These outcomes have the potential to enhance our understanding of EBV's involvement in periodontitis and advance periodontal research.

Antimicrobials offered from nature: Peroxidase-catalyzed systems and their mimics.

The control of antimicrobial resistance requires the development of novel antimicrobial alternatives and naturally occurring peroxidase-catalyzed systems may be of great value in this era of emerging antimicrobial resistance. In the peroxidase system, a peroxidase enzyme catalyzes the oxidation of a halide/pseudohalide, at the expense of hydrogen peroxide, to generate reactive products with broad antimicrobial properties. The appropriate use of peroxidase systems needs a better understanding of the identities and properties of the generated antimicrobial oxidants, specific targets in bacterial cells, their mode of action and the factors favoring or limiting their activity. Here, the ABCs (antibacterial activity, bacterial "backtalk" and cytotoxicity) of these systems and their mimics are discussed. Particular attention is paid to the concomitant use of thiocyanate and iodide dual substrates in peroxidase/peroxidase-free systems with implications on their antimicrobial activity. This review also provides a summary of actual applications of peroxidase systems as bio-preservatives in oral healthcare, milk industry, food/feed specialties and related products, mastitis and wound treatment; lastly, this review points to opportunities for further research and potential applications.

Epstein-Barr virus-infected plasma cells in periodontitis lesions.

Growing evidence supports that the Epstein-Barr virus (EBV) is a putative periodontal pathogen, but little is known regarding EBV behavior in periodontitis. Here, EBV infection was monitored in saliva and periodontal pocket (PP), at baseline and 3 months after periodontal non-surgical therapy (p-NST) in 20 patients diagnosed with periodontitis. After the treatment, the patients with the improved periodontal condition (good responders) showed a significant decrease in salivary EBV load. In contrast, in poor responders, EBV load was slightly increased. Moreover, after the therapy, most patients showed clear signs of EBV infection in a deep PP (≥5 mm) selected as a study site. To investigate how EBV can persist in a PP, we further investigate cellular sites of viral replication in PP. We identified large amounts of infiltrated EBV-infected cells, mostly overlapping with CD138+ plasma cells (PC). EBV-infected PCs formed high-density clusters within the infiltrate and along the periodontal epithelium which were commonly associated with CD3+ T-cells and CD20+ B-cells to evoke diffuse ectopic lymphoid-like structures. Taking together, this study provides new insights to support a model where the periodontal condition may play a major role in oral EBV shedding. Since PC harbors the late productive phases of EBV replication, the periodontal condition may favor B-cell differentiation with possible amplification of periodontal EBV infection and viral spreading. PCs have long been recognized as pathogenic markers in inflammatory lesions. Our finding sheds new light on the role of EBV infection and PC in periodontitis.

Construction and Validation of A Low-cost, Small-scale, Multiplex Continuous Culturing System for Microorganisms.

This protocol details the construction of a simple, low-cost, small-scale, multiplex chemostat system designed for the continuous cultivation of microorganisms in suspension (i.e., bacteria, yeast, microalgae). The continuous culture device can operate at a working volume of 25 ml and allows the run of 8 chemostats in parallel by a single person. It provides a platform for parallel, long-term studies of evolution and adaptation of microorganisms under the stress of antimicrobial agents and/or toxic pollutants. The system complies with the varied needs of researchers for an accessible, highly-throughput and reliable tool that is nevertheless easy to construct, use and operate, and not demanding of space, materials, medium supply and workload. Here, we also validate the use of this system to generate de novo resistance towards a novel antimicrobial and a commonly used antibiotic in an antimicrobial-sensitive model organism. We believe that this "Do It Yourself" (DIY) system may constitute a useful tool to address the global problem of antibiotic resistance and to develop non-antibiotic based therapies.

Detection of Epstein-Barr Virus in Periodontitis: A Review of Methodological Approaches.

Periodontitis, an inflammatory condition that affects the structures surrounding the tooth eventually leading to tooth loss, is one of the two biggest threats to oral health. Beyond oral health, it is associated with systemic diseases and even with cancer risk. Obviously, periodontitis represents a major global health problem with significant social and economic impact. Recently, a new paradigm was proposed in the etiopathogenesis of periodontitis involving a herpesviral-bacterial combination to promote long-term chronic inflammatory disease. Periodontitis as a risk factor for other systemic diseases can also be better explained based on viral-bacterial etiology. Significant efforts have brought numerous advances in revealing the links between periodontitis and Epstein-Barr virus (EBV), a gamma herpesvirus ubiquitous in the adult human population. The strong evidence from these studies may contribute to the advancement of periodontitis research and the ultimate control of the disease. Advancing the periodontitis research will require implementing suitable methods to establish EBV involvement in periodontitis. This review evaluates and summarizes the existing methods that allow the detection and diagnosis of EBV in periodontitis (also applicable in a more general way to other EBV-related diseases), and discusses the feasibility of the application of innovative emerging technologies.

Mutation rate and efflux response of bacteria exposed to a novel antimicrobial iodo-thiocyanate complex.

OBJECTIVES: Antimicrobials, at sub-lethal concentrations, can act as selectors and promoters of resistance by increasing mutation rates. We measured the rate of Escherichia coli mutation from levofloxacin (LVX) sensitivity to resistance when it was grown under the near-lethal challenge of the novel biocidal iodo-thiocyanate complex (ITC). Another relevant factor affecting the emergence of antimicrobial resistance is the role of efflux pumps. Consequently, we evaluated whether ITC could potentially be a substrate for efflux pumps, and thus that efflux-mediated resistance could arise towards ITC. METHODS: The mutation rate was measured by fluctuation analysis, when multiple parallel E. coli cultures were grown in the absence and presence of ITC. Then the mutational events, which occurred independently in each culture, were scored by plating the fraction of the culture in LVX-selective solid media and compared with the total cell number. To detect if ITC is a substrate for efflux pumps, minimum inhibitory concentrations (MICs) were determined against Pseudomonas aeruginosa in the absence and presence of the efflux pump inhibitor (EPI). RESULTS: We have found that the E. coli exposed to the near-lethal level of ITC had a slight, but not significant, increase in mutation rate compared with unexposed cultures. Furthermore, the presence of EPI decreased the MIC of ITC by a modest 2-fold, showing that ITC was not a target for efflux pumps. CONCLUSIONS: ITC usage most likely will not promote resistance development via increased mutation rates, and efflux-mediated resistance emergence to it is less likely than for some other antimicrobials.

New Viral Facets in Oral Diseases: The EBV Paradox.

The oral cavity contributes to overall health, psychosocial well-being and quality of human life. Oral inflammatory diseases represent a major global health problem with significant social and economic impact. The development of effective therapies, therefore, requires deeper insights into the etiopathogenesis of oral diseases. Epstein-Barr virus (EBV) infection results in a life-long persistence of the virus in the host and has been associated with numerous oral inflammatory diseases including oral lichen planus (OLP), periodontal disease and Sjogren's syndrome (SS). There is considerable evidence that the EBV infection is a strong risk factor for the development and progression of these conditions, but is EBV a true pathogen? This long-standing EBV paradox yet needs to be solved. This review discusses novel viral aspects of the etiopathogenesis of non-tumorigenic diseases in the oral cavity, in particular, the contribution of EBV in OLP, periodontitis and SS, the tropism of EBV infection, the major players involved in the etiopathogenic mechanisms and emerging contribution of EBV-pathogenic bacteria bidirectional interaction. It also proposes the involvement of EBV-infected plasma cells in the development and progression of oral inflammatory diseases. A new direction for preventing and treating these conditions may focus on controlling pathogenic EBV with anti-herpetic drugs.

Continuous culture of Escherichia coli, under selective pressure by a novel antimicrobial complex, does not result in development of resistance.

We attempted to generate de novo resistance to a newly described biocidal complex, ITC (iodo-thiocyanate complex), and to levofloxacin (LVX) in Escherichia coli ATCC 25922, by means of selective chemostat culture. We measured resistance by determining the minimum inhibitory concentrations (MICs) for these agents. E. coli underwent 20-day parallel adaptive evolution routes under no antimicrobial selection, and gradually increasing ITC and LVX selection pressure. Long-term exposure of E. coli to ITC did not induce resistance to ITC, or cross-resistance to LVX. No distinct mutational pattern was evidenced from whole-genome sequence (WGS)-based comparisons of ITC-challenged and unchallenged bacterial populations. Moreover, the exposed E. coli population could not survive a 2 × MIC challenge of ITC. By contrast, resistance to LVX was rapidly induced (on day 1 the MIC had increased 16-fold), selected for (by day 14 the MIC had increased 64-fold) and enriched with a highly characteristic genome mutational pattern. WGS of this evolving population revealed that the majority of mutations appeared in the genes of LVX target proteins (GyrA, ParC, ParE) and drug influx (OmpF). This study suggests that the usage of ITC may not trigger the emergence of facile resistance or cross-resistance, in contrast to common antibiotics.

In vitro comparative cytotoxicity study of a novel biocidal iodo-thiocyanate complex.

Novel biocides, which avoid the induction of cross-resistance to antibiotics, are an urgent societal requirement. Here, we compared the cytotoxic and bactericidal effects of a new antimicrobial agent, the iodo-thiocyanate complex (ITC), with those of the common antiseptics, hydrogen peroxide (H2O2), povidone iodine (PVP-I) and Lugol's iodine (Lugol). The antimicrobials were co-incubated for 10 min with HeLa and Escherichia coli cells in the presence and absence of organic matter (Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum). The cytotoxic concentrations of ITC were equivalent to its bactericidal concentrations (7.8 µg ml-1). By contrast, cytotoxic effects of H2O2, PVP-I and Lugol were apparent at concentrations lower than their bactericidal concentrations (250, 250 and 125 µg ml-1, respectively). The cellular effects of ITC were not quenched by organic matter, unlike the other antiseptics. ITC, PVP-I and Lugol had hemolytic effect on horse erythrocytes at high concentrations, while H2O2 showed no hemolysis. ITC, at 30 or 300 µg ml-1, did not cause DNA breakage in HeLa cells as assessed by an in vitro comet assay in the absence of S9 metabolic activation, whereas H2O2 caused extensive single-strand DNA breaks. The pronounced antimicrobial potency of ITC and its favorable cytotoxicity profile suggests that ITC should be considered for antiseptic applications.

Antibacterial Potential of an Antimicrobial Agent Inspired by Peroxidase-Catalyzed Systems.

Antibiotic resistance is an increasingly serious threat to global health. Consequently, the development of non-antibiotic based therapies and disinfectants, which avoid induction of resistance, or cross-resistance, is of high priority. We report the synthesis of a biocidal complex, which is produced by the reaction between ionic oxidizable salts-iodide and thiocyanate-in the presence of hydrogen peroxide as an oxidation source. The reaction generates bactericidal reactive oxygen and iodine species. In this study, we report that the iodo-thiocyanate complex (ITC) is an effective bactericidal agent with activity against planktonic and biofilm cells of Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus and methicillin-resistant S. aureus) bacteria. The minimum bactericidal concentrations and the minimum biofilm eradication concentrations of the biocidal composite were in the range of 7.8-31.3 and 31.3-250 µg ml-1, respectively. As a result, the complex was capable to cause a rapid cell death of planktonic test cultures at between 0.5 and 2 h, and complete eradication of dual and mono-species biofilms between 30 s and 10 min. Furthermore, the test bacteria, including a MRSA strain, exposed to the cocktail failed to develop resistance after serial passages. The antimicrobial activity of the ITC appears to derive from the combinational effect of the powerful species capable of oxidizing the essential biomolecules of bacteria. The use of this composition may provide an effective and efficient method for killing potential pathogens, as well as for disinfecting and removing biofilm contamination.

The role of stress in colicin regulation.

Bacteriocins produced by Enterobacteriaceae are high molecular weight toxic proteins that kill target cells through a variety of mechanisms, including pore formation and nucleic acid degradation. What is remarkable about these toxins is that their expression results in death to the producing cells and therefore bacteriocin induction have to be tightly regulated, often confined to times of stress. Information on the regulation of bacteriocins produced by enteric bacteria is sketchy as their expression has only been elucidated in a handful of bacteria. Here, we review the known regulatory mechanisms of enteric bacteriocins and explore the expression of 12 of them in response to various triggers: DNA-damaging agents, stringent response, catabolite repression, oxidative stress, growth phase, osmolarity, cold shock, nutrient deprivation, anaerobiosis and pH stress. Our results indicate that the expression of bacteriocins is mostly confined to mutagenic triggers, while all other triggers tested are limited inducers.