Nigella sativa essential oil and its bioactive compounds as resistance modifiers against Staphylococcus aureus.

Nigella sativa essential oil (EO) and its compounds (thymoquinone, carvacrol, and p-cymene) have a broad antimicrobial spectrum. The aim of this study was to investigate the antimicrobial and resistance modifying activity of N. sativa EO, thymoquinone, carvacrol, and p-cymene against one methicillin susceptible and one methicillin resistant Staphylococcus aureus strain. N. sativa EO, thymoquinone, carvacrol, and p-cymene were assessed for antimicrobial activity and modulation of antimicrobial resistance (by broth microdilution), inhibition of antimicrobial efflux (by ethidium bromide [EtBr] accumulation assay), relative expression of mepA gene (by real-time reverse transcriptase quantitative polymerase chain reaction), membrane disrupting effect (by LIVE/DEAD BacLight™ Kit), and finally antibiofilm activity (by the crystal violet assay). Both strains of S. aureus were susceptible to N. sativa EO, thymoquinone, and carvacrol. N. sativa EO and carvacrol induced the increase of EtBr accumulated by both S. aureus strains. Membrane integrity of ATCC strain was disrupted by carvacrol and p-cymene, whereas for the methicillin resistant S. aureus (MRSA) strain the membrane integrity was disrupted by each compound. N. sativa EO and its bioactive compounds such as carvacrol and p-cymene could be applied as resistance modifiers in MRSA strains.

Antiviral, Antimicrobial and Antibiofilm Activity of Selenoesters and Selenoanhydrides.

Selenoesters and the selenium isostere of phthalic anhydride are bioactive selenium compounds with a reported promising activity in cancer, both due to their cytotoxicity and capacity to reverse multidrug resistance. Herein we evaluate the antiviral, the biofilm inhibitory, the antibacterial and the antifungal activities of these compounds. The selenoanhydride and 7 out of the 10 selenoesters were especially potent antiviral agents in Vero cells infected with herpes simplex virus-2 (HSV-2). In addition, the tested selenium derivatives showed interesting antibiofilm activity against Staphylococcus aureus and Salmonella enterica serovar Typhimurium, as well as a moderate antifungal activity in resistant strains of Candida spp. They were inactive against anaerobes, which may indicate that the mechanism of action of these derivatives depends on the presence of oxygen. The capacity to inhibit the bacterial biofilm can be of particular interest in the treatment of nosocomial infections and in the coating of surfaces of prostheses. Finally, the potent antiviral activity observed converts these selenium derivatives into promising antiviral agents with potential medical applications.

Selenocompounds as Novel Antibacterial Agents and Bacterial Efflux Pump Inhibitors.

Bacterial multidrug resistance is becoming a growing problem for public health, due to the development and spreading of bacterial strains resistant to antimicrobials. In this study, the antibacterial and multidrug resistance reversing activity of a series of seleno-carbonyl compounds has been evaluated. The effects of eleven selenocompounds on bacterial growth were evaluated in Staphylococcus aureus, methicillin resistant S. aureus (MRSA), Enterococcus faecalis, Escherichia coli, and Chlamydia trachomatis D. The combination effect of compounds with antibiotics was examined by the minimum inhibitory concentration reduction assay. Their efflux pump (EP) inhibitory properties were assessed using real-time fluorimetry. Relative expressions of EP and quorum-sensing genes were studied by quantitative PCR. Results showed that a methylketone selenoester had remarkable antibacterial activity against Gram-positive bacteria and potentiated the activity of oxacillin in MRSA. Most of the selenocompounds showed significant anti-chlamydial effects. The selenoanhydride and the diselenodiester were active inhibitors of the AcrAB-TolC system. Based on these results it can be concluded that this group of selenocompounds can be attractive potential antibacterials and EP inhibitors. The discovery of new derivatives with a significant antibacterial activity as novel selenocompounds, is of high impact in the fight against resistant pathogens.

Expression of Chlamydia muridarum plasmid genes and immunogenicity of pGP3 and pGP4 in different mouse strains.

Chlamydia muridarum carries a cryptic plasmid (pMoPn) of 7.5kb, which encodes seven genes. Our aims were to describe the transcriptional pattern of the pMoPn genes in C. muridarum-infected mice and to evaluate the host immune responses against pGP3 and pGP4 proteins. BALB/c and C57BL/6N female mice were inoculated intranasally with C. muridarum and sacrificed at different time points, and the total RNA was extracted from the lung suspensions to determine the levels of expression of the different plasmid genes by RT qPCR. The supernatants of the lungs were subjected to the quantitation of recoverable C. muridarum. TCA04 and TCA05, which encode pGP3 and pGP4, respectively, were amplified by PCR and cloned into the pET vector. The proteins were overexpressed in E. coli HB101 and purified. Selected groups of BALB/c and C57BL/6N mice were infected with C. muridarum 1-3 times. The humoral immune responses in the sera of the mice to the proteins encoded by TCA04 and TCA05 were tested by Western blotting, and the cellular immune responses were assessed in lymphocyte proliferation assays. The proteins recognized by the mouse sera were further analysed by a LC/MSMS technique. The kinetics of C. muridarum growth were similar in the mouse strains used, but the pathogen burden was higher in the BALB/c mice in the late phase of infection. All the plasmid genes in the BALB/c mice showed an increased level of expression on day 7, whereas the expression of the same genes did not change on day 7 in the C57BL/6N mice. The levels of expression of the plasmid genes were higher in the C57BL/6N mice at later time points. In Western blot assays, the sera of the singly infected C57BL/6N mice reacted with the monomeric form of pGP3, whereas the sera of the singly infected BALB/c mice reacted with the trimeric form of pGP3. The sera of the multiply infected C57BL/6N mice also recognized pGP4. Similarly to the humoral immune response, cellular immune responses to pGP3 and pGP4 were detected in the C. muridarum-infected C57BL/6N mice, but the spleen cells of BALB/c mice responded with proliferation only to the pGP3 protein. These results suggest that the proteins encoded by pMoPn genes may modulate the host immune response during C. muridarum infection, and that the evolved immune response against plasmid proteins, similarly to that against other chlamydial proteins, depends on the genetic background of the host.

Chlamydophila pneumoniae re-infection triggers the production of IL-17A and IL-17E, important regulators of airway inflammation.

OBJECTIVE: Investigation of the effects of interleukin (IL)-17 cytokines in Chlamydophila pneumoniae-infected mice. METHODS: Mice were infected with C. pneumoniae once or three times and the expression of IL-17 cytokines was followed by RT qPCR from day 1 to day 28 after infection and re-infection. After the treatment of mice with anti-IL-17A, ELISA was used to detect the differences in cytokine and chemokine production. The number and phenotype of the IL-17A-producing cells were determined by ELISPOT. RESULTS: Chlamydophila pneumoniae induced IL-17A and IL-17F from day 2 after infection, and their levels remained elevated on day 28. The expression of IL-17C, IL-17D and IL-17E mRNA did not change significantly in response to a single infection. The in vivo neutralization of IL-17A resulted in a higher C. pneumoniae burden in the mouse lungs, a decreased cell influx, and diminished chemokine levels. The phenotype of IL-17A-producing cells was CD4(+). The re-infection of mice led to an increased expression of IL-17E mRNA. CONCLUSION: These results facilitate an understanding of the early inflammatory response after C. pneumoniae infection and suggest that C. pneumoniae re-infection induces the production of a high amount of IL-17E, which has an important role in the pathogenesis of allergic pulmonary diseases.

Inhibition of Bacterial Biofilm Formation by Phytotherapeutics with Focus on Overcoming Antimicrobial Resistance.

Bacteria within biofilms are more resistant to antibiotics and chemical agents than planktonic bacteria in suspension. Treatment of biofilm-associated infections inevitably involves high dosages and prolonged courses of antimicrobial agents; therefore, there is a potential risk of the development of antimicrobial resistance (AMR). Due to the high prevalence of AMR and its association with biofilm formation, investigation of more effective anti-biofilm agents is required. From ancient times, herbs and spices have been used to preserve foods, and their antimicrobial, anti-biofilm and anti-quorum sensing properties are well known. Moreover, phytochemicals exert their anti-biofilm properties at sub-inhibitory concentrations without providing the opportunity for the emergence of resistant bacteria or harming the host microbiota. With increasing scientific attention to natural phytotherapeutic agents, numerous experimental investigations have been conducted in recent years. The present paper aims to review the articles published in the last decade in order to summarize a) our current understanding of AMR in correlation with biofilm formation and b) the evidence of phytotherapeutic agents against bacterial biofilms and their mechanisms of action. The main focus has been put on herbal anti-biofilm compounds tested to date in association with Staphylococcus aureus, Pseudomonas aeruginosa and food-borne pathogens (Salmonella spp., Campylobacter spp., Listeria monocytogenes and Escherichia coli).

Chlamydia pneumoniae Influence on Cytokine Production in Steroid-Resistant and Steroid-Sensitive Asthmatics.

Medications for asthma management consisting of inhaled corticosteroids act by controlling symptoms. However, some patients do not respond to steroid treatment due to immunological factors at the cytokine level. Chlamydia pneumoniae (C. pneumoniae) infection is strongly implicated in asthma pathogenesis, causing altered immune responses. We investigated the association of C. pneumoniae serostatus with the production of certain cytokines by peripheral blood mononuclear cells (PBMCs) of steroid-resistant and -sensitive asthmatic patients. Our most important findings are the following: In the case of C. pneumoniae seropositive patients we detected pronounced spontaneous interleukin (IL)-10 secretion and, in the case of steroid-resistant patients, IL-10 secretion was at a significantly higher level as compared with in-sensitive patients (p < 0.01). Furthermore, steroid-resistant seropositive patients produced a significantly higher level of IL-10 spontaneously and under antigen stimulation as compared with steroid-resistant seronegative individuals (p < 0.05). Concerning spontaneous TNF-α secretion by C. pneumoniae seropositive asthmatics, we observed that steroid-resistant patients produced significantly more of this cytokine than steroid-sensitive patients. In the steroid-resistant patients' sera, a remarkably high MMP-9 concentration was associated with C. pneumoniae seronegativity. Our study revealed that the differences in the cytokine production in steroid-sensitive and -resistant asthmatic patients can be influenced by their C. pneumoniae serostatus.

Bioactive Compounds of Nigella Sativa Essential Oil as Antibacterial Agents against Chlamydia Trachomatis D.

Urogenital tract infection caused by obligate intracellular bacterium Chlamydia trachomatis D (CtrD) is a leading cause of sexually transmitted diseases. Essential oil (EO) of Nigella sativa has a broad antimicrobial spectrum. The aim of this study was to evaluate the antimicrobial activity of the bioactive compounds (p-cymene, thymoquinone, carvacrol, and thymol) of N. sativa EO against CtrD. The cytotoxic effects of the compounds were determined by MTT assay. In order to quantify the anti-chlamydial activity of the compounds, HeLa cells were infected with CtrD or CtrD treated previously with the compounds. The titer of the infectious CtrD was determined by indirect immunofluorescence assay. The minimum inhibitory concentrations of the compounds were evaluated by direct quantitative PCR. None of the compounds showed a cytotoxic effect on HeLa cells in the concentrations tested. According to the immunofluorescence assay, all of the compounds significantly inhibited the growth of CtrD. The quantitative PCR revealed that the minimum concentration that exerted anti-chlamydial activity was 3.12 µM in the case of thymoquinone and p-cymene, while that of carvacrol and thymol was 6.25 µM. Therefore, it can be concluded that bioactive compounds of N. sativa EO could be used as effective antimicrobial agents against CtrD.

The Opposite Effects of Kynurenic Acid and Different Kynurenic Acid Analogs on Tumor Necrosis Factor-α (TNF-α) Production and Tumor Necrosis Factor-Stimulated Gene-6 (TSG-6) Expression.

Purpose: The investigation of anti-inflammatory and immunosuppressive functions of Kynurenic acid (KYNA) is now in focus. There is also substantial evidence that TSG-6 has an anti-inflammatory activity. Therefore, in the present study, we compared the effects of newly synthetized KYNA analogs on the TNF-α production in U-937 monocytic cells in correlation with the effects on the TSG-6 expression. Methods: TNF-α production was measured by ELISA, the TSG-6 expression was determined by RTqPCR method. As cytokine inducers Staphylococcus aureus and Chlamydia pneumoniae were used. Results: KYNA and KYNA analogs attenuated TNF-α production and increased TSG-6 mRNA expression in U-937 cells stimulated by heat inactivated Staphylococcus aureus. In contrast, KYNA and some of the KYNA analogs increased the TNF-α production of C. pneumoniae infected U-937 cells; however, the newly synthetized analogs (SZR104, SZR 105, and SZR 109) exerted significant inhibitory effects on the TNF-α synthesis. The inhibitory and stimulatory effects correlated inversely with the TSG-6 expression. Conclusions: TSG-6 expression following activation with bacterial components could participate in the suppression of inflammatory cytokines, such as TNF-α, We suppose that the elevation of the TSG-6 expression by KYNA and especially by new KYNA analogs might be one of the mechanisms that are responsible for their suppressive effect on TNF-α production as a feedback mechanism. KYNA and KYNA analogs have an important role in influencing TSG-6 expression, and there is a possible benefit of targeting TSG-6 expression by kynurenines in inflammatory conditions following infections.

N-acetyl-cysteine increases the replication of Chlamydia pneumoniae and prolongs the clearance of the pathogen from mice.

Purpose. Within the community, 10 % of acquired pneumonia is caused by Chlamydia pneumoniae. N-acetyl-cysteine (NAC) is one of the most commonly used mucolytics in respiratory diseases, but its effect on C. pneumoniae infection has not yet been investigated. In this study, our aim was to investigate whether NAC influences the replication of C. pneumoniae. After determining that NAC does have an effect on C. pneumoniae replication, the effect of an alternative drug called Ambroxol (Ax) was investigated.Methodology. The in vitro effect of NAC and Ax was studied on C. pneumoniae-infected A549 and McCoy cells. Furthermore, the influence of NAC and Ax was examined in mice infected intranasally with C. pneumoniae.Results. NAC treatment resulted in approximately sixfold more efficient C. pneumoniae growth in tissue culture compared to the untreated control cells, and this effect was shown to be based on the increased binding of the bacterium to the host cells. The C. pneumoniae-infected mice to which NAC was given had prolonged and more severe infections than the control mice. Ax decreased C. pneumoniae replication in vitro, which was partially associated with the increased expression of indolamine 2,3-dioxygenase. In animals, using the adapted usual human dose, Ax did not alter the number of recoverable C. pneumoniae.Conclusion. Based on our results, it might be recommended that a mucolytic agent other than NAC, such as Ax, be used in respiratory diseases suspected to be caused by C. pneumoniae.

Growth characteristics of Chlamydia trachomatis in human intestinal epithelial Caco-2 cells.

Chlamydia trachomatis is an obligate intracellular bacterium causing infections of the eyes, urogenital and respiratory tracts. Asymptomatic, repeat and chronic infections with C. trachomatis are common in the urogenital tract potentially causing severe reproductive pathology. Animal models of infection and epidemiological studies suggested the gastrointestinal tract as a reservoir of chlamydiae and as a source of repeat urogenital infections. Thus, we investigated the growth characteristics of C. trachomatis in human intestinal epithelial Caco-2 cells and the infection-induced defensin production. Immunofluorescence staining and transmission electron microscopy showed the presence of chlamydial inclusions in the cells. Chlamydial DNA and viable C. trachomatis were recovered from Caco-2 cells in similar quantity compared to that detected in the usual in vitro host cell of this bacterium. The kinetics of expression of selected C. trachomatis genes in Caco-2 cells indicated prolonged replication with persisting high expression level of late genes and of heat shock protein gene groEL. Replication of C. trachomatis induced moderate level of ß-defensin-2 production by Caco-2 cells, which might contribute to avoidance of immune recognition in the intestine. According to our results, Caco-2 cells support C. trachomatis replication, suggesting that the gastrointestinal tract is a site of residence for these bacteria.

Chlamydia pneumoniae Infection Exacerbates Atherosclerosis in ApoB100only/LDLR-/- Mouse Strain.

AIMS: Hyperlipidaemia model animals have been used to elucidate the role of Chlamydia pneumoniae (Cpn) infection in atherosclerosis. The aims of this study were to investigate the proatherogenic effect of multiple Cpn infections in ApoB100only/LDLR-/- mice which based on lipid profile can be regarded as the most suitable mouse model of human hypercholesterolemia and to compare the lesion development to that in a major atherosclerosis model ApoE-/- mice. METHODS AND RESULTS: Aorta samples of ApoB100only/LDLR-/- mice infected three times with Cpn were subjected to morphometric analyses. Morphometric evaluation disclosed that Cpn infections exacerbated atherosclerosis development in the aortic root and descending aorta of the mice fed with normal diet. Viable Cpn was detected in the ascending aorta by RT-PCR. Chlamydial 16SrRNA expression showed the presence of viable Cpn in the aorta of infected animals. A similar rate of acceleration of atherosclerosis was observed when the infection protocol was applied in ApoB100only/LDLR-/- and in ApoE-/- mice. CONCLUSION: Similar to ApoE-/- mice, ApoB100only/LDLR-/- mice with more human-relevant serum lipoprotein composition develop increased atherosclerosis after Cpn infections; thus this mouse strain can be used as a model of infection-related atherosclerosis enhancement and can provide further evidence for the proatherogenic influence of Cpn in mice.

A direct quantitative PCR-based measurement of herpes simplex virus susceptibility to antiviral drugs and neutralizing antibodies.

Herpes simplex viruses (HSV) are common human pathogens that can cause painful but benign manifestations and recurrent complaints, but can also cause significant morbidity and mortality on infection of the eye or brain and with disseminated infection of an immunosuppressed patient or a neonate. HSV growth inhibition measurement by plaque or yield reduction is a key task in the development of novel antiviral compounds but the manual methods are very labour intensive. The sensitive and specific PCR technology could be an effective method for quantitation of HSV DNA related to virus replication; however the currently described PCR approaches have a major limitation, namely the requirement of purification of DNA from the infected cells. This limitation makes this approach unfeasible for high-throughput screenings. The monitoring of HSV specific antibody titre is essential in vaccination trials and in the improvement of HSV-based oncolytic virotherapy. Usually, conventional cytopathic effect-based and plaque reduction neutralization tests are applied to measure the neutralization titre, but these methods are also time-consuming. To overcome this, we developed a quantitative PCR (qPCR) method for the detection of HSV-2 DNA directly from the infected cells (direct qPCR) and the method was further adapted to measure the titre of HSV specific neutralizing antibody in human sera. The conditions of direct qPCR assay were optimized to measure the antiviral activity of known and novel antiviral substances. Using HSV-2 seronegative and seropositive patients' sera, the validity of the direct qPCR neutralization test was compared to traditional cytopathic effect-based assay. The direct qPCR method was able to detect the HSV-2 DNA quantitatively between multiplicity of infection 1/64 and 1/4194304, indicating that the dynamic range of the detection was approximately 65,500 fold with high correlation between the biological and technical replicates. As a proof of the adaptability of the method, we applied the direct qPCR for antiviral inhibitory concentration 50 (IC50) measurements of known and novel antiviral compounds. The measured IC50 of acyclovir was ∼0.28µg/ml, similar to the previously published IC50 value. The IC50 of novel antiviral candidates was between 1.6-3.1µg/ml. The direct qPCR-based neutralization titres of HSV positive sera were 1:32-1:64, identical to the neutralization titres determined using a traditional neutralization assay. The negative sera did not inhibit the HSV-2 replication in either of the tests. Our direct qPCR method for the HSV-2 growth determination of antiviral IC50 and neutralization titre is less time-consuming, less subjective and a more accurate alternative to the traditional plaque titration and growth reduction assays.

Protection promoted by pGP3 or pGP4 against Chlamydia muridarum is mediated by CD4(+) cells in C57BL/6N mice.

Urogenital tract infection with Chlamydia trachomatis is a leading cause of sexually transmitted infections. There is currently no commercially available vaccine against C. trachomatis. The highly conserved plasmid of chlamydiae has been considered to be a virulence factor and the plasmid proteins have important roles in the Chlamydia-specific immune response. This study was designed to evaluate the efficacy of vaccination with plasmid proteins in the prevention of C. muridarum lung infection in a mouse model. C57BL/6N mice were immunised 3 times subcutaneously with recombinant pGP3 or pGP4 and infected with C. muridarum. Immunisation of the mice with recombinant pGP3 or pGP4 protein caused a significantly lower chlamydial burden in the lungs of the infected mice; the lower IFN-γ level indicated a reduced extent of inflammation. In vitro or in vivo neutralisation of C. muridarum with sera obtained from immunised mice did not reduce the number of viable C. muridarum in the lungs of mice. However, adoptive transfer of the CD4(+) spleen cells isolated from the immunised mice resulted in a significantly reduced bacterial burden. Our results indicate that it is not the pGP3- and pGP4-specific antibodies, but the CD4(+) cells that are responsible for the protective effect of the immune response to plasmid proteins.

Anti-chlamydial effect of plant peptides.

Even in asymptomatic cases of Chlamydia trachomatis infection, the aim of the antibiotic strategy is eradication of the pathogen so as to avoid the severe late sequelae, such as pelvic inflammatory disease, ectopic pregnancy, and tubal infertility. Although first-line antimicrobial agents have been demonstrated to be predominantly successful in the treatment of C. trachomatis infection, treatment failures have been observed in some cases. Rich source of antimicrobial peptides was recently discovered in Medicago species, which act in plants as differentiation factors of the endosymbiotic bacterium partner. Several of these symbiotic plant peptides have proved to be potent killers of various bacteria in vitro. We show here that 7 of 11 peptides tested exhibited antimicrobial activity against C. trachomatis D, and that the killing activity of these peptides is most likely due to their interaction with specific bacterial targets.

IL-17E production is elevated in the lungs of Balb/c mice in the later stages of Chlamydia muridarum infection and re-infection.

BACKGROUND: Pathogens can influence allergic respiratory diseases. We previously found that multiple infections with Chlamydophila pneumoniae induce the production of interleukin-17A (IL-17A) and IL-17E, which have roles in the pathogenesis of asthma. The present work was designed to investigate our hypothesis that infections with another pathogen can induce the production of IL-17A and IL-17E. MATERIALS AND METHODS: At an internal of 28 days, mice were infected twice with Chlamydia muridarum; the kinetics of IL-17A and IL-17E expression was subsequently determined at the mRNA and protein levels. The amounts of IL-17 cytokines produced by the stimulated spleen cells were determined by enzyme-linked immunosorbent assay (ELISA). The presence of IL-17E in the lungs was revealed by an indirect immunofluorescence test. RESULTS: The infection with C. muridarum induced the production of IL-17A at the early stages of infection. The quantity of IL-17E was highest on days 28 and 56 after the first infection (28 days after the second infection). In the later stages of infection, IL-17E was produced by epithelial cells. The re-stimulated peripheral spleen cells produced IL-17A. CONCLUSION: Multiple infection with C. muridarum induces the production of a high amount of IL-17E, which plays an important part in the pathogenesis of allergic pulmonary diseases.