The Role of Quorum Sensing in Antimicrobial Action

“Quorum sensing” - yeterli çoğunluğu algılama, bakteri hücrelerinin eş güdüm içinde hareket ederek, çok hücreli organizma özelliği göstermesidir. Hücreler arası sinyal iletimi, kültür biyokitlesinin kontrolü, biyofilm oluşumu, motilite ve virülans genlerinin ifadelenmesi gibi birçok olay üzerinde etkilidir. Bakteriyel direnme (persistans), antimikrobiyal baskısı altında ölen hücrelerin arasından ne ölen ne de hızlı üreyen bireylerin çıkması olarak tanımlanmaktadır. Başta biyofilm enfeksiyonları olmak üzere, antimikrobiyaller ile tedavi edilemeyen enfeksiyonların çoğundan direnen hücreler sorumlu tutulmaktadır. Bu çalışmada, hücreler arası iletişimden sorumlu üniversal otoindükleyici, otoindükleyici 2\'nin, Escherichia coli hücrelerinde ampisiline direnme fenotipinin ortaya çıkmasındaki rolü araştırılmıştır. Bu amaçla, rekombinant DNA teknikleri kullanılarak Escherichia coli K12 DH5α suşunun luxS pozitif ve negatif izogenik mutantları oluşturulmuştur. Bu mutantlara ampisilin etkisi altında zamana karşı öldürme deneyi yapılmıştır. ampisilin etkisi altındaki luxS pozitif ve negatif bakteri kültürlerindeki hücre sayısındaki azalmanın 120. dakikaya kadar paralel gittiği gözlenmiştir. Bu noktadan sonra canlı kalan luxS negatif bakteri sayısında keskin bir düşüş gözlenmiştir. Sonuç olarak, bu çalışmada, “quorum sensing”in önemli aracılarından olan otoindükleyici 2 üretiminin antimikrobiyal baskısı altında direnen hücre fenotipinin oluşumunda önemli olduğu gösterilmiştir.Quorum sensing is the coordinated behaviour of bacterial cells to act like a multicellular organism. Cell to cell signaling has impact on the control of culture biomass, biofilm formation, motility and the expression of the virulence genes. Bacterial persistence is the emergence of a neither dying nor actively dividing population of cells amoung dying cells in the presence of antimicrobials. These persisters are considered responsible for treatment failures in infectious diseases such as biofilm infections. In this study, the role of universal autoinducer, autoinducer-2 (AI-2) in persistence of Escherichia coli cells in the presence of ampicillin has been investigated. For this purpose, recombinant DNA techniques was used to construct luxS positive and negative isogenic mutants of E. coli K12 DH5α. Time kill assay was performed by using these mutants in the presence of ampicillin. After the addition of ampicillin to the media, the decrease in live cell mass was similar in luxS positive and negative bacterial cultures until the end of 120 minutes. Beyond this time point, a sharp decrease in surviving luxS negative bacteria. As a result, AI-2, an important mediator of quorum sensing, is suggested to play an important role in bacterial persistence in the presence of antimicrobial agents

Impact of glutamine on the effect of neopterin in methyl mercury-exposed neurons

Exposure to methyl mercury (MeHg), induces blood-brain barrier damage leading to non-selective influx of cytotoxic agents, besides the entrance of inflammatory cells into the brain. However, there is no data available regarding the effects of co-treatment of neopterin and interferon-gamma (IFN-gamma) in MeHgexposed SH-SY5Y dopaminergic neurons. MeHg-exposed SH-SY5Y human neuroblastoma cells were treated with neopterin and IFN-gamma in the presence and absence of L-Glutamine. Cell viability was determined by MTT assay. Oxidative stress intensity coefficient was calculated by taking into consideration the amount of nitric oxide production per viable neuron. 5μM MeHg was found to be more toxic than 1μM or 2μM doses of MeHg for SH-SY5Y cells in glutamine-containing medium. Furthermore, 0.1μM neopterin supplementation significantly increased the neuronal cell viability while, oxidative stress significantly decreased. Glutamine supplementation in culture medium, not only enhanced the MeHg toxicity, but also supported the antioxidant effect of neopterin. These results indicate that neopterin has a protective effect on MeHg toxicity in SH-SY5Y neurons. Neopterin was more effective in improving the total mitochondrial metabolic activity of cells exposed to 5μM MeHg in comparison to IFN-gamma. Although IFN-gamma supplementation alone partially improved 5μM MeHg toxicity on neurons, it weakened the protective effect of neopterin

Impact of glutamine on the effect of neopterin in methyl mercury-exposed neurons

Exposure to methyl mercury (MeHg), induces blood-brain barrier damage leading to non-selective influx of cytotoxic agents, besides the entrance of inflammatory cells into the brain. However, there is no data available regarding the effects of co-treatment of neopterin and interferon-gamma (IFN-gamma) in MeHgexposed SH-SY5Y dopaminergic neurons. MeHg- exposed SH-SY5Y human neuroblastoma cells were treated with neopterin and IFN-gamma in the presence and absence of L-Glutamine. Cell viability was determined by MTT assay. Oxidative stress intensity coefficient was calculated by taking into consideration the amount of nitric oxide production per viable neuron. 5 mu M MeHg was found to be more toxic than 1 mu M or 2 mu M doses of MeHg for SH-SY5Y cells in glutamine-containing medium. Furthermore, 0.1 mu M neopterin supplementation significantly increased the neuronal cell viability while, oxidative stress significantly decreased. Glutamine supplementation in culture medium, not only enhanced the MeHg toxicity, but also supported the antioxidant effect of neopterin. These results indicate that neopterin has a protective effect on MeHg toxicity in SH-SY5Y neurons. Neopterin was more effective in improving the total mitochondrial metabolic activity of cells exposed to 5 mu M MeHg in comparison to IFN-gamma. Although IFN- gamma supplementation alone partially improved 5 mu M MeHg toxicity on neurons, it weakened the protective effect of neopterin

CARACTERIZACIÓN Y EXTRACCIÓN DE LA LISOZIMA DE LA CLARA DE HUEVO

Lysozyme is a small protein (14kDa) that occurs in almost all body fluids, and tissues of animal organisms. It exhibits bacteriolytic activity due to its ability of breaking bacterial cell walls. The demand of this enzyme has increased because of its diverse uses in pharmacy or food industry. Different methods for isolation have been proposed. Most of them are used in laboratory practice to obtain the pure enzyme of high activity, however only some of these methods are feasible on a commercial scale. The most useful method of lysozyme extraction are chromatography tecnhniques. Other methods, such as crystallization, aqueous two-phase extraction or membrane filtration, have also been reported. In this work, we studied a two-step protocol for the lysozyme extraction from chicken egg white, based on a protein precipitation coupled with chromatography. Three precipitation agents (polyethilenglycol, ammonium sulfate and sodium chloride) and four types of chromatography (cation-exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography and affinity chromatography) were assayed to determine the efficiency of each methodology. The most efficient methodology was the combination of protein precipitation using 3% polyethilenglycol and ion exchange chromatography, obtaining lysozyme of great purity and the activity was as high as 72 U/m

Two important controversial risk factors in SARS-CoV-2 infection: Obesity and smoking

The effects of obesity and smoking in the coronavirus disease 2019 (COVID-19) pandemic remain controversial. Angiotensin converting enzyme 2 (ACE2), a component of the renin-angiotensin system (RAS), is the human cell receptor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19. ACE2 expression increases on lung alveolar epithelial cells and adipose tissue due to obesity, smoking and air pollution. A significant relationship exists between air pollution and SARS-CoV-2 infection, as more severe COVID-19 symptoms occur in smokers; comorbid conditions due to obesity or excess ectopic fat accumulation as underlying risk factors for severe COVID-19 strongly encourage the virus/ACE2 receptor-ligand interaction concept. Indeed, obesity, air pollution and smoking associated risk factors share underlying pathophysiologies that are related to the Renin-Angiotensin-System in SARS-CoV-2 infection. The aim of this review is to emphasize the mechanism of receptor-ligand interaction and its impact on the enhanced risk of death due to SARS-CoV-2 infection

Glutamate‑mediated effects of caffeine and interferon‑γ on mercury-induced toxicity.

The molecular mechanisms mediating mercury‑induced neurotoxicity are not yet completely understood. Thus, the aim of this study was to investigate whether the severity of MeHg‑ and HgCl2‑mediated cytotoxicity to SH‑SY5Y human dopaminergic neurons can be attenuated by regulating glutamate‑mediated signal‑transmission through caffeine and interferon‑γ (IFN‑γ). The SH‑SY5Y cells were exposed to 1, 2 and 5 µM of either MeHgCl2 or HgCl2 in the presence or absence of L‑glutamine. To examine the effect of adenosine receptor antagonist, the cells were treated with 10 and 20 µM caffeine. The total mitochondrial metabolic activity and oxidative stress intensity coefficient were determined in the 1 ng/ml IFN‑γ‑ and glutamate‑stimulated SH‑SY5Y cells. Following exposure to mercury, the concentration‑dependent decrease in mitochondrial metabolic activity inversely correlated with oxidative stress intensity. MeHg was more toxic than HgCl2. Mercury‑induced neuronal death was dependent on glutamate‑mediated excitotoxicity. Caffeine reduced the mercury‑induced oxidative stress in glutamine-containing medium. IFN‑γ treatment decreased cell viability and increased oxidative stress in glutamine‑free medium, despite caffeine supplementation. Although caffeine exerted a protective effect against MeHg-induced toxicity with glutamate transmission, under co‑stimulation with glutamine and IFN‑γ, caffeine decreased the MeHg‑induced average oxidative stress only by half. Thereby, our data indicate that the IFN‑γ stimulation of mercury‑exposed dopaminergic neurons in neuroinflammatory diseases may diminish the neuroprotective effects of caffeine

Glutamate-mediated effects of caffeine and interferon-gamma on mercury-induced toxicity

The molecular mechanisms mediating mercury-induced neurotoxicity are not yet completely understood. Thus, the aim of this study was to investigate whether the severity of MeHg-and HgCl2-mediated cytotoxicity to SH-SY5Y human dopaminergic neurons can be attenuated by regulating glutamate-mediated signal-transmission through caffeine and interferon-gamma (IFN-gamma). The SH-SY5Y cells were exposed to 1, 2 and 5 mu M of either MeHgCl2 or HgCl2 in the presence or absence of L-glutamine. To examine the effect of adenosine receptor antagonist, the cells were treated with 10 and 20 mu M caffeine. The total mitochondrial metabolic activity and oxidative stress intensity coefficient were determined in the 1 ng/ml IFN-gamma-and glutamate-stimulated SH-SY5Y cells. Following exposure to mercury, the concentration-dependent decrease in mitochondrial metabolic activity inversely correlated with oxidative stress intensity. MeHg was more toxic than HgCl2. Mercury-induced neuronal death was dependent on glutamate-mediated excitotoxicity. Caffeine reduced the mercury-induced oxidative stress in glutaminecontaining medium. IFN-gamma treatment decreased cell viability and increased oxidative stress in glutamine-free medium, despite caffeine supplementation. Although caffeine exerted a protective effect against MeHg-induced toxicity with glutamate transmission, under co-stimulation with glutamine and IFN-gamma, caffeine decreased the MeHg-induced average oxidative stress only by half. Thereby, our data indicate that the IFN-gamma stimulation of mercury-exposed dopaminergic neurons in neuroinflammatory diseases may diminish the neuroprotective effects of caffeine

Assessment of the Immunogenicity and Protective Aspects of a DNA Vaccine Targeting Crimean Congo Hemorrhagic Fever Virus Glycoprotein Gc

Aim: Crimean Congo Hemorrhagic Fever (CCHF) is a lethal, endemic infectious disease inhuman. For the preventive measures of the disease, there is currently no safe and efficient vaccine,widely for human use. Vaccine development for CCHF virus is an actively researched subject. Inthis study, we aimed to investigate the immunizing and protective potentials of the CCHF virussurface glycoprotein Gc that is delivered as a single antigen via a DNA based vaccine vector.Material and Methods: A DNA based vaccine targeting the immunogenic envelope glycoproteinGc of a CCHF virus isolate with Turkey origin (Ank2) was generated and its immunogenicity andprotective capability against lethal challenge in IFN?/?R-/- receptor knock out mice was assessed.Results: The developed vaccine candidate (pGc) elicited a considerable amount of neutralizingantibody responses in the vaccinated mice. The vaccine candidate significantly induced bothantiviral Th1 and B cell activating Th2 immune responses deduced from the cytokineproduction profiles in the vaccinated mice. However, despite the immune responses elicitedpost-immunization, the vaccine failed to confer protection against lethal CCHF virus infection.Conclusion: To the best of our knowledge, this is the first report of a DNA vaccine candidategenerated against CCHF virus based on the glycoprotein Gc. The pGc vaccine candidate exhibitedantigen-specific immunity in IFN/?/?R-/- mice, but was unable to produce a protection upon lethalchallenge with the homologous CCHF virus. Once we comprehensively understand the immunecorrelates of protection, we will be more eligible to significantly improve the efficacy of vaccines

Assessment of the immunogenicity and protective aspects of a dna vaccine targeting crimean congo hemorrhagic fever virus glycoprotein gc Kırım kongo kanamalı ateşi virüsü glikoprotein gc’yi hedef alan bir dna aşısının bağışıklık ve koruyuculuk sağlama özelliklerinin değerlendirilmesi

Aim: Crimean Congo Hemorrhagic Fever (CCHF) is a lethal, endemic infectious disease inhuman. For the preventive measures of the disease, there is currently no safe and efficient vaccine,widely for human use. Vaccine development for CCHF virus is an actively researched subject. Inthis study, we aimed to investigate the immunizing and protective potentials of the CCHF virussurface glycoprotein Gc that is delivered as a single antigen via a DNA based vaccine vector.Material and Methods: A DNA based vaccine targeting the immunogenic envelope glycoproteinGc of a CCHF virus isolate with Turkey origin (Ank2) was generated and its immunogenicity andprotective capability against lethal challenge in IFN?/?R-/- receptor knock out mice was assessed.Results: The developed vaccine candidate (pGc) elicited a considerable amount of neutralizingantibody responses in the vaccinated mice. The vaccine candidate significantly induced bothantiviral Th1 and B cell activating Th2 immune responses deduced from the cytokineproduction profiles in the vaccinated mice. However, despite the immune responses elicitedpost-immunization, the vaccine failed to confer protection against lethal CCHF virus infection.Conclusion: To the best of our knowledge, this is the first report of a DNA vaccine candidategenerated against CCHF virus based on the glycoprotein Gc. The pGc vaccine candidate exhibitedantigen-specific immunity in IFN/?/?R-/- mice, but was unable to produce a protection upon lethalchallenge with the homologous CCHF virus. Once we comprehensively understand the immunecorrelates of protection, we will be more eligible to significantly improve the efficacy of vaccines

Investigation of the Presence of Disinfectant Resistance Genes qacA/B in Nosocomial Methicillin-Resistant Staphylococcus aureus Isolates and Evaluation of Their In Vitro Disinfectant Susceptibilities

Development of resistance to disinfectant substances in nosocomial microorganisms is an important problem encountered during disinfectant practices. Methicillin-resistant Staphylococcus aureus (MRSA) remains a significant cause of hospital-acquired infections. Besides being resistant to several antimicrobial agents, MRSA strains can also become resistant to some disinfectant substances. Resistance to disinfectant substances may develop due to the misuse of disinfectants. This may either be due to the frequent use of disinfectant substances or use in lower concentrations than recommended. MRSA strains may harbour the qacA/B disinfectant resistance genes that may cause resistance to quarternary ammonium compounds and some cationic disinfectants. These resistance genes are found in plasmids and are responsible for decreased susceptibility or resistance. In this study, a total of 69 nosocomial MRSA strains isolated from clinical specimens in our hospital were tested for disinfectant activity and the presence of qacA/B disinfectant resistance genes in these isolates was investigated by polymerase chain reaction. We determined whether the presence of these genes caused phenotypic resistance to chlorhexidine and benzalkonium chloride by the use of bactericidal and bacteriostatic tests. For this purpose, the minimum inhibitory concentration (MIC) values of these disinfectants against MRSA isolates were detected by microdilution method with the proposals of CLSI, and bactericidal effects of these disinfectants were also detected by using quantitative suspension test according to EN13727:2003 European Standard. It has been found that 11.6% (8/69) of the isolates harbored qacA/B resistance genes. MIC values for chlorhexidine and benzalkonium chloride were found in the range of 2-8 mu g/ml. Although it was observed that MIC values were higher in five of the qacA/B gene positive isolates, statistically significant difference was not found between gene positive and gene negative groups. Both 1% chlorhexidine and 1% benzalkonium chloride were found bactericidal against the isolates including the ones carrying the qacA/B resistance genes. It was concluded that the presence of the qacA/B disinfectant resistance genes did not lead to resistance to the disinfectant substances at the concentrations used in clinical practices. Furthermore, tested disinfectants still exhibited bactericidal activity even with high MIC values