Adenosine A2A receptor as a potential regulator of Mycobacterium leprae survival mechanisms: new insights into leprosy neural damage.

Background: Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which can lead to a disabling neurodegenerative condition. M. leprae preferentially infects skin macrophages and Schwann cells-glial cells of the peripheral nervous system. The infection modifies the host cell lipid metabolism, subverting it in favor of the formation of cholesterol-rich lipid droplets (LD) that are essential for bacterial survival. Although researchers have made progress in understanding leprosy pathogenesis, many aspects of the molecular and cellular mechanisms of host-pathogen interaction still require clarification. The purinergic system utilizes extracellular ATP and adenosine as critical signaling molecules and plays several roles in pathophysiological processes. Furthermore, nucleoside surface receptors such as the adenosine receptor A2AR involved in neuroimmune response, lipid metabolism, and neuron-glia interaction are targets for the treatment of different diseases. Despite the importance of this system, nothing has been described about its role in leprosy, particularly adenosinergic signaling (AdoS) during M. leprae-Schwann cell interaction. Methods: M. leprae was purified from the hind footpad of athymic nu/nu mice. ST88-14 human cells were infected with M. leprae in the presence or absence of specific agonists or antagonists of AdoS. Enzymatic activity assays, fluorescence microscopy, Western blotting, and RT-qPCR analysis were performed. M. leprae viability was investigated by RT-qPCR, and cytokines were evaluated by enzyme-linked immunosorbent assay. Results: We demonstrated that M. leprae-infected Schwann cells upregulated CD73 and ADA and downregulated A2AR expression and the phosphorylation of the transcription factor CREB (p-CREB). On the other hand, activation of A2AR with its selective agonist, CGS21680, resulted in: 1) reduced lipid droplets accumulation and pro-lipogenic gene expression; 2) reduced production of IL-6 and IL-8; 3) reduced intracellular M. leprae viability; 4) increased levels of p-CREB. Conclusion: These findings suggest the involvement of the AdoS in leprosy neuropathogenesis and support the idea that M. leprae, by downmodulating the expression and activity of A2AR in Schwann cells, decreases A2AR downstream signaling, contributing to the maintenance of LD accumulation and intracellular viability of the bacillus.

Adenosine A2a receptor as a potential regulator of Mycobacterium leprae survival mechanisms: new insights into leprosy neural damage

Background: Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which can lead to a disabling neurodegenerative condition. M. leprae preferentially infects skin macrophages and Schwann cells­glial cells of the peripheral nervous system. The infection modifies the host cell lipid metabolism, subverting it in favor of the formation of cholesterol-rich lipid droplets (LD) that are essential for bacterial survival. Although researchers have made progress in understanding leprosy pathogenesis, many aspects of the molecular and cellular mechanisms of host­pathogen interaction still require clarification. The purinergic system utilizes extracellular ATP and adenosine as critical signaling molecules and plays several roles in pathophysiological processes. Furthermore, nucleoside surface receptors such as the adenosine receptor A2AR involved in neuroimmune response, lipid metabolism, and neuron­glia interaction are targets for the treatment of different diseases. Despite the importance of this system, nothing has been described about its role in leprosy, particularly adenosinergic signaling (AdoS) during M. leprae­Schwann cell interaction. Methods: M. leprae was purified from the hind footpad of athymic nu/nu mice. ST88-14 human cells were infected with M. leprae in the presence or absence of specific agonists or antagonists of AdoS. nzymatic activity assays, fluorescence microscopy, Western blotting, and RT-qPCR nalysis were performed. M. leprae viability was investigated by RT-qPCR, and cytokines were evaluated by enzymelinked immunosorbent assay. Results: We demonstrated that M. leprae-infected Schwann cells upregulated CD73 and ADA and downregulated A2AR expression and the phosphorylation of the transcription factor CREB (p-CREB). On the other hand, activation of A2AR with its selective agonist, CGS21680, resulted in: 1) reduced lipid droplets accumulation and pro-lipogenic gene expression; 2) reduced production of IL-6 and IL-8; 3) reduced intracellular M. leprae viability; 4) increased levels of p-CREB. Conclusion: These findings suggest the involvement of the AdoS in leprosy neuropathogenesis and support the idea that M. leprae, by downmodulating the expression and activity of A2AR in Schwann cells, decreases A2AR downstream signaling, contributing to the maintenance of LD accumulation and intracellular viability of the bacillus.

A PMAxxTM qPCR Assay Reveals That Dietary Administration of the Microalgae Tetraselmis chuii Does Not Affect Salmonella Infantis Caecal Content in Early-Treated Broiler Chickens.

Salmonella enterica serovars cause infections in humans. S. enterica subsp. enterica serovar Infantis is considered relevant and is commonly reported in poultry products. Evaluating innovative approaches for resisting colonization in animals could contribute to the goal of reducing potential human infections. Microalgae represent a source of molecules associated with performance and health improvement in chickens. Tetraselmis chuii synthesizes fermentable polysaccharides as part of their cell wall content; these sugars are known for influencing caecal bacterial diversity. We hypothesized if its dietary administration could exert a positive effect on caecal microbiota in favor of a reduced S. Infantis load. A total of 72 one-day-old broiler chickens (COBB 500) were randomly allocated into three groups: a control, a group infected with bacteria (day 4), and a group challenged with S. Infantis but fed a microalgae-based diet. Caecal samples (n = 8) were collected two days post-infection. A PMAxxTM-based qPCR approach was developed to assess differences regarding bacterial viable load between groups. The inclusion of the microalga did not modify S. Infantis content, although the assay proved to be efficient, sensitive, and repeatable. The utilized scheme could serve as a foundation for developing novel PCR-based methodologies for estimating Salmonella colonization.

Behavior of Salmonella Enteritidis and Shigella flexneri during induction and recovery of the viable but nonculturable state.

Bacteria may enter into a viable but nonculturable (VBNC) state as a response to stresses, such as those found in food processing. Cells in the VBNC state lose the ability to grow in a conventional culture medium but man recover culturability. The viability, culturability and intracellular reactive oxygen species (ROS) of Salmonella Enteritidis and Shigella flexneri were evaluated under stress conditions to induce a VBNC state. Cells were maintained under nutritional, osmotic and cold stresses (long-term induction) in Butterfield's phosphate solution plus 1.2 M of NaCl at 4°C and under nutritional and oxidative stresses (short-term induction) in 10 mM of H2O2. Culture media, recovery agents, sterilization methods of media and incubation temperature, were combined and applied to recover the culturability of the VBNC cells. Salmonella entered in the VBNC state after 135 days under long-term induction, while Shigella maintained culturability after 240 days. Under short-term induction, Salmonella and Shigella lose culturability after 135 and 240 min, respectively. Flow cytometric analysis revealed viable cells and intracellular ROS in both species in VBNC. It was not possible to recover the culturability of VBNC cells using the 42 combinations of different factors.

How Functionalized Surfaces Can Inhibit Bacterial Adhesion and Viability.

Device-associated infections (DAI) remain a serious concern in modern healthcare. Bacterial attachment to a surface is the first step in biofilm formation, which is one of the main causes of DAIs. The development of materials capable of preventing or inhibiting bacterial attachment constitutes a promising approach to deal with this problem. The multifactorial nature of biofilm maturation and antibiotic resistance directs the research for multitargeted or combinatorial therapeutic approaches. One attractive strategy is the modification or the engineering of surfaces in order to provide antiadhesive and/or antimicrobial properties. Currently, several different approaches that involve physical and chemical surface modification deliver some possible alternatives to achieve this goal. The engineered surfaces can be coated with molecules capable of inhibiting the bacterial adhesion or with active agents that kill microorganisms. In addition, surfaces can also be modified in order to be stimuli-responsive, responding to a particular trigger and then delivering the consequent antimicrobial outcome. Here, we review the prevailing strategies to modify surfaces in order to create an antimicrobial surface and discuss how different surface functionalization can affect bacterial adhesion and/or viability.

High frequency equipment promotes antibacterial effects dependent on intensity and exposure time.

BACKGROUND: The indiscriminate use of antibiotics has caused bacteria to develop mechanisms of resistance to antibacterial agents, limiting treatment options. Therefore, there is a great need for alternative methods to control infections, especially those related to skin. One of the alternative methods is the high frequency equipment (HFE), which is used on skin conditions as an analgesic, an anti-inflammatory, and mainly to accelerate cicatricial processes and have a bactericidal effect through the formation of ozone. This research investigated the antibacterial effect of HFE on standard cultures of bacteria. MATERIALS AND METHODS: Dilutions (104 colony forming unit mL-1) were performed for Enterobacter aerogenes and Staphylococcus aureus with 24-hour growth bacteria. Then, 1 µL of each dilution was pipetted into suitable medium and the HFE flashing technique was used at intensities of 6, 8 and 10 mA for 30, 60, 90, 120 and 180 seconds. The control group received no treatment. Plates were incubated at 37°C for 24 hours and then read. RESULTS: The spark at intensity of 6 mA had no bactericidal effect on the E. aerogenes; however, a significant bacterial growth reduction occurred at intensity of 8 mA after 120 and 180 seconds, and at 10 mA, reduction in bacterial growth could already be verified at 30 seconds and total bacterial growth inhibition occurred in 180 seconds. For S. aureus, there was a strong bacterial growth inhibition at all intensities used; however, at 6 mA, absence of bacterium growth after 120 and 180 seconds was observed. By increasing the flashing intensity to 8 and 10 mA, it was observed that the bacterium growth was inhibited after only 30 seconds of irradiation. CONCLUSION: The HFE has time-dependent antibacterial effects against E. aerogenes and S. aureus bacteria that have several resistance mechanisms.

Prokaryotic RNA Associated to Bacterial Viability Induces Polymorphonuclear Neutrophil Activation.

Polymorphonuclear neutrophils (PMN) are the first cellular line of antibacterial host defense. They sense pathogens through recognition of pathogen-associated molecular patterns (PAMPs) by innate pattern recognition receptors, such as Toll-like receptors (TLR). The aim of this study was to investigate whether PMN sense bacterial viability and explore which viability factor could be involved in this phenomenon. For this purpose, different functions were evaluated in isolated human PMN using live Escherichia coli (Ec) and heat-killed Ec (HK-Ec). We found that bacterial viability was indispensable to induce PMN activation, as measured by forward-scatter (FSC) increase, CD11b surface expression, chemotaxis, reactive oxygen species (ROS) generation and neutrophil extracellular trap (NET) formation. As uncapped non-polyadenylated prokaryotic mRNA has been recognized as a PAMP associated to bacterial viability by macrophages and dendritic cells, total prokaryotic RNA (pRNA) from live Ec was purified and used as a stimulus for PMN. pRNA triggered similar responses to those observed with live bacteria. No RNA could be isolated from HK-Ec, explaining the lack of effect of dead bacteria. Moreover, the supernatant of dead bacteria was able to induce PMN activation, and this was associated with the presence of pRNA in this supernatant, which is released in the killing process. The induction of bactericidal functions (ROS and NETosis) by pRNA were abolished when the supernatant of dead bacteria or isolated pRNA were treated with RNAse. Moreover, endocytosis was necessary for pRNA-induced ROS generation and NETosis, and priming was required for the induction of pRNA-induced ROS in whole blood. However, responses related to movement and degranulation (FSC increase, CD11b up-regulation, and chemotaxis) were still triggered when pRNA was digested with RNase, and were not dependent on pRNA endocytosis or PMN priming. In conclusion, our results indicate that PMN sense live bacteria through recognition of pRNA, and this sensing triggers potent bactericidal mechanisms.

Viability and Effects on Bacterial Proteins by Oral Rinses with Hypochlorous Acid as Active Ingredient

Abstract: This study investigated the effect of hypochlorous acid (HOCl) rinses and chlorhexidine (CHX) on the bacterial viability of S. mutans, A. israelii, P. gingivalis, A. actinomycetemcomitans, E. corrodens, C. rectus, K. oxytoca, K. pneumoniae and E. cloacae. The percentage of live bacteria was tested by fluorescence method using Live/Dead kit(r) and BacLight (Molecular Probes(r)) and compared between groups by the Kruskal-Wallis and U Mann-Whitney tests with Bonferroni correction (p value<0.012). The effect of HOCl and CHX on total proteins of P. gingivalis and S. mutans was determined by SDS-PAGE. CHX showed a higher efficacy than HOCl against S. mutans, A. israelii, E. corrodens and E. cloacae (p<0.001) while HOCl was more effective than CHX against P. gingivalis, A. actinomycetemcomitans, C. rectus and K. oxytoca (p=0.001). CHX and HOCl had similar efficacy against K. pneumoniae. Proteins of P. gingivalis and S. mutans were affected similarly by HOCl and CHX. HOCl reduced the bacterial viability especially in periodontopathic bacteria, which may support its use in the control of subgingival biofilm in periodontal patients.

Resumo: Este estudo investigou o efeito de enxaguantes à base de ácido hipocloroso (HOCl) e clorexidina (CHX) sobre a viabilidade bacteriana de S. mutans, A. israelii, P. gingivalis, A. actinomycetemcomitans, E. corrodens, C. rectus, K. oxytoca, K. pneumoniae e E. cloacae. O percentual de bactérias sobreviventes foi testado pelo método de fluorescência utilizando Live/Dead kit(r) e BacLight (Molecular Probes(r)), fazendo comparação entre os grupos com os testes de Kruskal-Wallis e U Mann-Whitney e correção de Bonferroni (p<0,012). O efeito de HOCl e CHX sobre P. gingivalis e S. mutans foi determinado por SDS-PAGE. O CHX mostrou eficácia superior ao HOCl contra S. mutans, A. israelii, E. corrodens e E. cloacae (p<0,001), ao passo que P. gingivalis, A. actinomycetemcomitans, C. rectus e K. oxytoca foram melhores que o CHX para o HOCl (p=0,001). O K. pneumoniae teve efeito similar para o CHX e para o HOCl. As proteínas de P. gingivalis e S. mutans foram afetadas de modo semelhante por CHX e HOCl. O HOCl reduziu a viabilidade bacterial, especialmente nas bactérias periodontopáticas, o que pode recomendar o uso no controle do biofilme subgingival em pacientes periodentais.