Silver nanoparticle with potential antimicrobial and antibiofilm efficiency against multiple drug resistant, extensive drug resistant Pseudomonas aeruginosa clinical isolates.

BACKGROUND: The study aims to investigate the effect of combining silver nanoparticles (AGNPs) with different antibiotics on multi-drug resistant (MDR) and extensively drug resistant (XDR) isolates of Pseudomonas aeruginosa (P. aeruginosa) and to investigate the mechanism of action of AGNPs. METHODS: AGNPs were prepared by reduction of silver nitrate using trisodium citrate and were characterized by transmission electron microscope (TEM) in addition to an assessment of cytotoxicity. Clinical isolates of P. aeruginosa were collected, and antimicrobial susceptibility was conducted. Multiple Antibiotic Resistance (MAR) index was calculated, and bacteria were categorized as MDR or XDR. Minimum inhibitory concentration (MIC) of gentamicin, ciprofloxacin, ceftazidime, and AGNPs were determined. The mechanism of action of AGNPs was researched by evaluating their effect on biofilm formation, swarming motility, protease, gelatinase, and pyocyanin production. Real-time PCR was performed to investigate the effect on the expression of genes encoding various virulence factors. RESULTS: TEM revealed the spherical shape of AGNPs with an average particle size of 10.84 ± 4.64 nm. AGNPS were safe, as indicated by IC50 (42.5 µg /ml). The greatest incidence of resistance was shown against ciprofloxacin which accounted for 43% of the bacterial isolates. Heterogonous resistance patterns were shown in 63 isolates out of the tested 107. The MAR indices ranged from 0.077 to 0.84. Out of 63 P. aeruginosa isolates, 12 and 13 were MDR and XDR, respectively. The MIC values of AGNPs ranged from 2.65 to 21.25 µg /ml. Combination of AGNPs with antibiotics reduced their MIC by 5-9, 2-9, and 3-10Fold in the case of gentamicin, ceftazidime, and ciprofloxacin, respectively, with synergism being evident. AGNPs produced significant inhibition of biofilm formation and decreased swarming motility, protease, gelatinase and pyocyanin production. PCR confirmed the finding, as shown by decreased expression of genes encoding various virulence factors. CONCLUSION: AGNPs augment gentamicin, ceftazidime, and ciprofloxacin against MDR and XDR Pseudomonas isolates. The efficacy of AGNPs can be attributed to their effect on the virulence factors of P. aeruginosa. The combination of AGNPs with antibiotics is a promising strategy to attack resistant isolates of P. aeruginosa.

Genetic diversity of macrolides resistant Staphylococcus aureus clinical isolates and the potential synergistic effect of vitamins, C and K3.

BACKGROUND: Macrolide antibiotics have been extensively used for the treatment of Staphylococcus aureus infections. However, the emergence of macrolide-resistant strains of S. aureus has become a major concern for public health. The molecular mechanisms underlying macrolide resistance in S. aureus are complex and diverse, involving both target site modification and efflux pump systems. In this study, we aim to overcome the molecular diversity of macrolide resistance mechanisms in S. aureus by identifying common molecular targets that could be exploited for the development of novel therapeutics. METHODS: About 300 Staphylococcus aureus different isolates were recovered and purified from 921 clinical specimen including urine (88), blood (156), sputum (264), nasal swabs (168), pus (181) and bone (39) collected from different departments in Tanta University Hospital. Macrolide resistant isolates were detected and tested for Multi Drug Resistant (MDR). Gel electrophoresis was performed after the D test and PCR reaction for erm(A), (B), (C), msr(A), and mph(C) genes. Finally, we tried different combinations of Erythromycin or Azithromycin antibiotics with either vitamin K3 or vitamin C. RESULTS: Macrolide resistance S. aureus isolates exhibited 7 major resistance patterns according to number of resistance markers and each pattern included sub patterns or subgroups. The PCR amplified products of different erm genes; analysis recorded different phenotypes of the Staphylococcus aureus isolates according to their different genotypes. In addition, our new tested combinations of Erythromycin and vitamin C, Erythromycin, and vitamin K3, Azithromycin and vitamin C and Azithromycin and vitamin K3 showed significant antibacterial effect when using every antibiotic alone. Our findings provide new insights into the molecular mechanisms of macrolide resistance in S. aureus and offer potential strategies for the development of novel protocols to overcome this emerging public health threat.

Pseudomonas aeruginosa's greenish-blue pigment pyocyanin: its production and biological activities.

A subject of great interest is the bioprospecting of microorganisms and their bioactive byproducts, such as pigments. Microbial pigments have various benefits, including being safe to use due to their natural makeup, having therapeutic effects, and being produced all year round, regardless of the weather or location. Pseudomonas aeruginosa produces phenazine pigments that are crucial for interactions between Pseudomonas species and other living things. Pyocyanin pigment, which is synthesized by 90-95% of P. aeruginosa, has potent antibacterial, antioxidant, and anticancer properties. Herein, we will concentrate on the production and extraction of pyocyanin pigment and its biological use in different areas of biotechnology, engineering, and biology.

Antibacterial, antibiofilm, and anti-quorum sensing activities of pyocyanin against methicillin-resistant Staphylococcus aureus: in vitro and in vivo study.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) infections are considered a major public health problem, as the treatment options are restricted. Biofilm formation and the quorum sensing (QS) system play a pivotal role in S. aureus pathogenicity. Hence, this study was performed to explore the antibacterial effect of pyocyanin (PCN) on MRSA as well as its effect on MRSA biofilm and QS. RESULTS: Data revealed that PCN exhibited strong antibacterial activity against all test MRSA isolates (n = 30) with a MIC value equal to 8 µg/ml. About 88% of MRSA biofilms were eradicated by PCN treatment using the crystal violet assay. The disruption of MRSA biofilm was confirmed using confocal laser scanning microscopy, which showed a reduction in bacterial viability (approximately equal to 82%) and biofilm thickness (approximately equal to 60%). Additionally, the disruption of the formation of microcolonies and the disturbance of the connection between bacterial cells in the MRSA biofilm after PCN treatment were examined by scanning electron microscopy. The 1/2 and 1/4 MICs of PCN exerted promising anti-QS activity without affecting bacterial viability; Agr QS-dependent virulence factors (hemolysin, protease, and motility), and the expression of agrA gene, decreased after PCN treatment. The in silico analysis confirmed the binding of PCN to the AgrA protein active site, which blocked its action. The in vivo study using the rat wound infection model confirmed the ability of PCN to modulate the biofilm and QS of MRSA isolates. CONCLUSION: The extracted PCN seems to be a good candidate for treating MRSA infection through biofilm eradication and Agr QS inhibition.

A purified and lyophilized Pseudomonas aeruginosa derived pyocyanin induces promising apoptotic and necrotic activities against MCF-7 human breast adenocarcinoma.

BACKGROUND: Pyocyanin, a specific extracellular secondary metabolite pigment produced by Pseudomonas aeruginosa, exhibits redox activity and has toxic effects on mammalian cells, making it a new and potent alternative for treating cancer. Breast cancer (BC) treatment is now defied by acquired and de novo resistance to chemotherapy, radiation, or targeted therapies. Therefore, the anticancer activity of purified and characterized pyocyanin was examined against BC in our study. RESULTS: The maximum production of pyocyanin (53 µg/ml) was achieved by incubation of the highest pyocyanin-producing P. aeruginosa strain (P32) in pH-adjusted peptone water supplemented with 3% cetrimide under shaking conditions at 37 °C for 3 days. The high purity of the extracted pyocyanin was proven by HPLC against standard pyocyanin. The stability of pyocyanin was affected by the solvent in which it was stored. Therefore, the purified pyocyanin extract was lyophilized to increase its shelf-life up to one year. Using the MTT assay, we reported, for the first time, the cytotoxic effect of pyocyanin against human breast adenocarcinoma (MCF-7) with IC50 = 15 µg/ml while it recorded a safe concentration against human peripheral blood mononuclear cells (PBMCs). The anticancer potential of pyocyanin against MCF-7 was associated with its apoptotic and necrotic activities which were confirmed qualitatively and quantitively using confocal laser scanning microscopy, inverted microscopy, and flow cytometry. Caspase-3 measurements, using real-time PCR and western blot, revealed that pyocyanin exerted its apoptotic activity against MCF-7 through caspase-3 activation. CONCLUSION: Our work demonstrated that pyocyanin may be an ideal anticancer candidate, specific to cancer cells, for treating MCF-7 by its necrotic and caspase-3-dependent apoptotic activities.