The promising anti-virulence activity of candesartan, domperidone, and miconazole on Staphylococcus aureus.

Staphylococcus aureus is a primary cause of hospital and community-acquired infections. With the emergence of multidrug-resistant S. aureus strains, there is a need for new drugs discovery. Due to the poor supply of new antimicrobials, targeting virulence of S. aureus may generate weaker selection for resistant strains, anti-virulence agents disarm the pathogen instead of killing it. In this study, the ability of the FDA-approved drugs domperidone, candesartan, and miconazole as inhibitors of S. aureus virulence was investigated. The effect of tested drugs was evaluated against biofilm formation, lipase, protease, hemolysin, and staphyloxanthin production by using phenotypic and genotypic methods. At sub-inhibitory concentrations, candesartan, domperidone, and miconazole showed a significant inhibition of hemolysin (75.8-96%), staphyloxanthin (81.2-85%), lipase (50-65%), protease (40-64%), and biofilm formation (71.4-90%). Domperidone and candesartan have similar activity and were more powerful than miconazole against S. aureus virulence. The hemolysins and lipase inhibition were the greatest under the domperidone effect. Candesartan showed a remarkable reduction in staphyloxanthin production. The highest inhibitory effect of proteolytic activity was obtained with domperidone and candesartan. Biofilm was significantly reduced by miconazole. Expression levels of crtM, sigB, sarA, agrA, hla, fnbA, and icaA genes were significantly reduced under candesartan (68.98-82.7%), domperidone (62.6-77.2%), and miconazole (32.96-52.6%) at sub-MIC concentrations. Candesartan showed the highest inhibition activity against crtM, sigB, sarA, agrA, hla, and icaA expression followed by domperidone then miconazole. Domperidone showed the highest downregulation activity against fnbA gene. In conclusion, candesartan, domperidone, and miconazole could serve as anti-virulence agents for attenuation of S. aureus pathogenicity.

Impeding Virulence of Candida albicans by Candesartan and Domperidone.

Candida albicans is the most common human fungal pathogen that has developed extensive virulence factors which allows successful colonization and infection of the host. Anti-virulence agents can alleviate the pathogenesis of fungi and help the immune system to eradicate them easily. This study aimed to explore the anti-virulence effect of domperidone and candesartan against C. albicans standard strain. Sub-inhibitory concentrations (1/4 and 1/8 of minimum inhibitory concentration) of domperidone and candesartan significantly inhibited the virulence factors hemolysin, lipase, protease, phospholipase, and bioflim formation. It was found that candesartan inhibited biofilm formation by 60.48-67.91%, hemolysin activity (61.21-74.14%), phospholipase activity (40-49.67%), lipase activity (58.97-73%), and protease activity (52.63%), while domperidone was found to inhibit biofilm formation by 70.54-77.49%, hemolysin activity (64.84-69.84%), phospholipase activity (49.67-60%), lipase activity (50-54.87%), and protease activity (52.63-57.9%). Quantitative real time-PCR confirmed the anti-virulence activity of domperidone and candesartan as both drugs significantly reduce the expression of the virulence genes SAP2, SAP6, PLB1, PLB2, LIP4, LIP5. In conclusion, domperidone and candesartan could serve as anti-virulence agents for treatment of C. albicans infections.

Diclofenac mitigates virulence of multidrug-resistant Staphylococcus aureus.

Staphylococcus aureus is an opportunistic pathogen that has the ability to cause a wide range of diseases including superficial infection and severe invasive life threatening infections. The pathogenicity of S. aureus is mediated by a group of virulence factors that mediate the colonization and penetration. The antibiotic resistance of S. aureus has evolved due to the abuse of antibiotics rendering the cure of infection very difficult especially with the shortage in new antibiotic production. To combat this shortage, repurposing of FDA-approved drugs against the virulence factors is a new strategy. The analgesic drug Diclofenac was found to have anti-virulence activity against Pseudomonas aeruginosa and Proteus mirabilis. This study aimed to demonstrate the anti-virulence effect of diclofenac against clinical MRSA isolates phenotypically and genotypically using qRT-PCR. In this study, diclofenac showed significant reduction in biofilm formation when compared to controls, the inhibition ranged between 22.67% and 70%. Also, remarkable inhibition of hemolysin activity was found (5.4-66.34%). Additionally, diclofenac has inhibitory activity against the staphyloxanthin production (8-57.2%). The results were confirmed by qRT-PCR that showed significant down-regulation of tested virulence genes. The down-regulation ranged from 43 to 64.05% for SarA, 36.85-64.75% for AgrA, 50-63.2% for hla, 38.55-60.35% for FnbA, 46.75-61.05% for IcaA, 27.55-64% for SigB and 51.05-72.8% for CrtM. In conclusion, diclofenac can be used in combination with antibiotics as anti-virulence agent against MDR-MRSA which will enhance the ability of immune system to eradicate infection.

Selective isolation of Avian influenza virus (AIV) from cloacal samples containing AIV and Newcastle disease virus.

Avian influenza viruses (AIVs) are important zoonotic pathogens whose natural reservoir is waterfowl. In addition to AIV, waterfowl are often coinfected with other viruses, such as the paramyxoviruses, of which Newcastle disease virus (NDV) is of particular importance because of the highly virulent nature of certain strains of this virus for domestic poultry. In routine surveillance of waterfowl for AIV, a number of cloacal samples were encountered that were positive for AIV by real-time reverse transcription polymerase chain reaction (RT-PCR), but did not yield AIV by inoculation in embryonated chicken eggs. On further testing, these samples were also positive for NDV by conventional RT-PCR. It was hypothesized that if both NDV and AIV are present in a sample, the former may overgrow AIV yielding false-negative AIV results. Such samples were treated with chicken anti-NDV polyclonal antiserum and then inoculated in embryonated chicken eggs. Several samples were found to be positive for different subtypes of AIV, indicating that, in the presence of mixed infection with NDV and AIV, it is imperative to remove the influence of NDV, so a true picture of AIV prevalence emerges. An additional benefit is that information on the circulation of NDV in these birds sheds light on their epidemiologic and ecologic significance.

Isolation of Avian influenza virus from polymerase chain reaction-negative cloacal samples of waterfowl.

Avian influenza virus (AIV) is one of the most important zoonotic pathogens because of its potential to cause severe disease outbreaks in avian and human hosts. Virus isolation in embryonated chicken eggs (ECEs) remains a gold standard technique for AIV detection. However, some laboratories prefer molecular methods, such as real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), for initial sample screening because of their high throughput sample processing and rapid results. Samples found positive on real-time qRT-PCR are then inoculated in ECEs for virus isolation and characterization. This approach is based on the premise that real-time qRT-PCR will detect all AIV-positive samples. The current study aimed to determine if AIV can be isolated from cloacal samples of waterfowl that were initially found to be negative by real-time qRT-PCR screening. Quantitative RT-PCR-negative cloacal samples (1,369) were inoculated for virus isolation in commercial nonspecific pathogen-free ECEs. After 4 days of incubation, the allantoic fluids were harvested and inoculated in fresh ECEs for a second passage. Allantoic fluids from 147 samples were positive for hemagglutination with chicken erythrocytes. Of the 147 hemagglutination-positive allantoic fluids, 82 were AIV positive when confirmed with real-time qRT-PCR. Ten isolates were subtyped as H7N2 (n  =  7), H7N1, H1N2, and H2N2. In addition, N subtype could be determined for isolates from an additional 25 samples. These results highlight the fact that screening by real-time qRT-PCR may result in some false-negative cloacal samples for AIV.

Triggered replenishment imaging reduces variability of quantitative myocardial contrast echocardiography and allows assessment of myocardial blood flow reserve.

Assessment of replenishment kinetics (RK) following ultrasound-induced destruction of contrast microbubbles allows quantification of myocardial blood flow reserve (MBFR) applying the model f (t) = A (1 - e(-betat)), with parameter beta describing mean flow velocity and parameter A representing blood volume. However, few data on the variability and reproducibility of RK in a clinical setting are available. Therefore, we examined 30 patients in a rest-adenosine protocol in one center. Off-line quantification of real-time perfusion imaging (RTPI) and triggered replenishment imaging (TRI) was performed at two sites and compared with coronary angiography and flow reserve measurements. Parameter A was found to be robust in all investigated segments (coefficient of variation (CV) < 7.2%+/- 5.1). Variability was lowest for parameter beta using TRI in apical segments (CV 6.5%+/- 5.2, P < 0.01). Highest CV was found with RTPI in lateral segments (CV : 39.8%+/- 40.6). Concerning day-to-day reproducibility both methods revealed similar results within range of heterogeneity of myocardial blood flow. Both sites obtained significantly lower MBFR in patients with flow-limiting CAD, compared to subjects without (P < 0.01). Correlation of both sites showed close relationship (y = 0.88x + 0.45, r = 0.83, P < 0.0001), without systematic bias. TRI significantly reduces variability of RK in quantitative MCE. Assessment of MBFR allows investigator-independent evaluation of CAD.