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.

Characterization and genomic analysis of novel bacteriophage NK20 to revert colistin resistance and combat pandrug-resistant Klebsiella pneumoniae in a rat respiratory infection model.

AIM: We investigated the therapeutic capacity of the isolated Klebsiella bacteriophage NK20 against pandrug-resistant strains. Moreover, we assessed the impact of resistance development on the overall therapeutic outcome both in vitro and in vivo. MAIN METHODS: The pandrug-resistant K. pneumoniae Kp20 is used as a host strain for the isolation of bacteriophages using sewage samples. Spot assay was then used to compare the spectra of the isolated phages, while kinetic and genomic analysis of the phage with the broadest spectrum was assessed. Antibacterial potential of the phage was assessed using turbidimetric assay and MIC with and without colistin. Finally, the therapeutic efficacy was evaluated in vivo using a rat respiratory infection model. KEY FINDINGS: The isolated lytic bacteriophage (NK20) showed a relatively broad spectrum and an acceptable genomic profile. In vitro antibacterial assay revealed bacterial resistance development after 12 h. Colistin inhibited bacterial regrowth and reduced pandrug-resistant strains' colistin MICs. Despite the isolation of resistant clones, intranasal administration of NK20 significantly (p < 0.05) reduced the bacterial load in both the pulmonary and blood compartments and rescued 100 % of challenged rats. Histological and immunological analysis of treated animals' lung tissue revealed less inflammation and lower TNF-α and caspase-3 expression. SIGNIFICANCE: NK20 is a promising candidate that rescued rats from untreatable, pan-drug-resistant K. pneumoniae Kp20. Moreover, it steers the evolution of resistant mutants with higher sensitivity to colistin and less virulence, opening the door for using phages as sensitizing and anti-virulence entities rather than direct killer.

Exploring the potential efficacy of phage therapy for biocontrol of foodborne pathogenic extensively drug-resistant Escherichia coli in gastrointestinal tract of rat model.

AIM: The emergence of extensively drug-resistant (XDR) Escherichia coli leaves little or no therapeutic options for the control of these foodborne pathogens. The goal is to isolate, characterize, and assess the potential efficacy of a bacteriophage in the treatment of an induced gastrointestinal tract infection. MAIN METHODS: Sewage water was used to isolate phage phPE42. Transmission electron microscope was used for the visualization of phage morphology. Lysis profile, growth kinetics, and stability studies were determined. The ability of phage to eradicate biofilms was assessed by crystal violet staining, resazurin assay, compound bright field microscope, and confocal laser scanning microscope (CLSM). Moreover, the efficacy of phage phPE42 as a potential therapy was evaluated in a rat model. KEY FINDINGS: A newly lytic Myoviridae phage phPE42 was isolated and exhibited broad coverage activity (48.6 %) against E. coli clinical isolates. It demonstrated favorable growth kinetics and relative stability under a variety of challenging conditions. The resazurin colorimetric assay and CLSM provided evidence of phage potential's ability to significantly (P < 0.05) decrease the viability of biofilm-embedded cells. The bacterial burden in animal faeces was effectively eradicated (P < 0.05) by oral administration of phage phPE42. Phage-treated rats exhibited a significant decrease in tissue damage with no signs of inflammation, necrosis, or erosion. Furthermore, phage therapy significantly (P < 0.05) reduced the expression level of the apoptotic marker caspase-3 and the inflammatory cytokine TNF-α. SIGNIFICANCE: Treatment with phage phPE42 is considered a promising alternative therapy for the control of severe foodborne infections spurred by pathogenic XDR E. coli.

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.

Differential Impact of Specific Amino Acid Residues on the Characteristics of Avian Influenza Viruses in Mammalian Systems.

Avian influenza virus (AIV) H9N2 was declared to be endemic in birds of the Middle East, in particular in Egypt, with multiple cases of human infections. Despite concerns about the pandemic threat posed by H9N2 AIV, due to the fact that its receptor specificity is similar to that of human influenza viruses, its morbidity and mortality rates in humans are so far negligible. However, the acquisition of specific adaptive amino acid (aa) mutations in the viral polymerase can enhance cross-species transmission of the virus itself or of reassortants, which gained these changes. The polymerase basic protein 2 (PB2) is one of the key determinants for AIV adaptation towards mammals. Although mammalian pathogenicity-related mutations (MPMs) in PB2 genes were identified in different AIVs, the specific effect of single or multiple mutations on viral fitness has not been compared so far. Here, we studied the effect of the aa K at position 591, which was frequently reported in the PB2 of Egyptian H9N2 isolates, on the proliferation efficiency and polymerase activity of an H5N1 (clade 2.2.1.2) AIV already carrying the mammalian adaptive mutation 627K. Using reverse genetics, we generated a set of recombinant parental strains and H5N1 variants carrying the avian-like 591Q/627E or mammalian-like adaptive mutations 591K/627K (H5N1EGY, H9N2EGY, H5N1PB2-H9N2EGY, H5N1H9N2_PB2_K591Q, H5N1PB2_K627E, H5N1PB2_K627E/591K, H5N1PB2_627K/591K). Regardless of the avian-like 627E or the mammalian-adaptive 627K, both variants carrying the 591K (H5N1PB2_K627E/591K, H5N1PB2_627K/591K) and the reassortant H5N1PB2-H9N2EGY replicated to significantly higher levels in mammalian continuous MDCK and Calu-3 cell lines and primary normal human bronchial epithelial cells than the parental H5N1EGY virus (carrying solely the 627K adaptive mutation). Expectedly, the H5N1 variants carrying avian-like PB2 mutations (H5N1H9N2_PB2_K591Q, H5N1PB2_K627E) replicated to significantly lower levels than the parental H5N1EGY virus in the predefined primary and continuous mammalian cell line systems. Consistently, the activity of H5N1 subtype AIV polymerase complexes comprising PB2 segments with singular 591K or combined with 627K was significantly enhanced when compared to parental H5N1EGY and H9N2EGY. This study emphasizes the significant impact of 591K containing PB2 segments in the background of H5N1 polymerase on viral fitness in addition to the well-known MPM 627K in vitro.

Characterization of newly isolated bacteriophage to control multi-drug resistant Pseudomonas aeruginosa colonizing incision wounds in a rat model: in vitro and in vivo approach.

AIMS: Pseudomonas aeruginosa is one of the most common causes of opportunistic and hospital-acquired infections in the world, which is repeatedly associated with treatment challenges. The evolution of new approaches such as phage therapy may be a novel alternative strategy for the treatment of these life-threatening infections. This paper aims to characterize the isolated bacteriophage and evaluate its potential therapy for the treatment of induced skin infection. MAIN METHODS: Enrichment method and double-layer overlay agar were used for isolation and purification of bacteriophages. The lysis profiles of isolated phages were evaluated using spot method. The phage morphology was visualized by transmission electron microscope. The growth kinetics such as adsorption rate, latent period, burst size, and in vitro challenging activity were determined. Biofilm eradication was analyzed using confocal laser scanning microscope (CLSM). Furthermore, the potential activity of phage therapy was evaluated in a rat model. KEY FINDINGS: Eight phages were isolated while phage phPS127 displayed the strongest lytic spectra. This phage is a member of Siphoviridae family that showed good growth kinetics. Our in vitro results showed that phage phPS127 significantly decreased the bacterial density (P < 0.05). CLSM revealed the significant reduction in the viability of the biofilm-adhered cells (P < 0.05). Phage therapy provided a significant level of treatment and promoted wound healing. Moreover, phage therapy significantly decreased bacterial burden (P < 0.05), inflammatory cytokine (TNF-α) and apoptosis (caspase-3) expression level. SIGNIFICANCE: Phage phPS127 can be considered as a promising candidate for treatment of clinical P. aeruginosa infections.

A comparison between collaborative and single surgeon approach in endoscopic endonasal surgery to sphenoid sinus.

PURPOSE: Endoscopic endonasal transsphenoidal surgery (EETS) requires abundant collaborative work between neurosurgeon and ear, nose, and throat (ENT) surgeon. In low-volume centers, however, the surgery may be carried out completely and solely by a neurosurgeon. The current study evaluates the differences in both technique and complications in the approach to the sphenoid sinus for endoscopic endonasal approach (EEA) performed solely by a single neurosurgeon compared to collaborative effort between neurosurgery and otolaryngology. METHODS: The study comprises 50 consecutive patients with intra-sellar pituitary lesions undergoing EETS. Half of the patients were operated completely by single neurosurgeon (group A) and the other half by collaboration between single ENT surgeon, as a primary surgeon during nasal step, and the neurosurgeon (group B). Both groups were assessed intra-operatively as to operative technique, average time of EEA to sphenoid sinus, and presence of endonasal structural difficulties and complications. RESULTS: A significant difference was recorded between both groups regarding average time of EEA to sphenoid sinus (P < 0.001) and incidence of intraoperative nasal complications (P = 0.006). There was a difference between ENT surgeon and neurosurgeon adopting the same approach to sphenoid sinus. Sphenoid sinus approaches from group B characterized by their short duration (mean 10 vs 22 min) and low incidence of intraoperative endonasal complications (4.8% vs 28%). CONCLUSION: Study results emphasized the necessity of collaboration between neurosurgeon and ENT surgeon in endoscopic endonasal approaches, to efficiently deal with intraoperative endonasal difficulties and complications which pose difference for both surgeons performing the same surgical procedure.

Optimization of niosomes for enhanced antibacterial activity and reduced bacterial resistance: in vitro and in vivo evaluation.

OBJECTIVE: The aim was to optimize norfloxacin niosomes for enhanced antibacterial activity and reduced bacterial resistance. METHODS: Pseudomonas aeruginosa, a biofilm forming bacterium, was used as the test organism. Different norfloxacin niosomes were evaluated in vitro and in vivo, respectively, for antibacterial activity compared with aqueous drug solution. The influence of norfloxacin niosomes on biofilm formation was investigated. The interaction of niosomes with bacterial cells was also monitored using the scanning electron microscopy (SEM). RESULTS: The efficacy of niosomes depended on their composition. Standard niosomes of Span 60 and cholesterol were similar to drug solution. Incorporation of Tween 80, oleic acid (OA), OA/propylene glycol or lecithin produced fluid niosomes which reduced the MIC and inhibited biofilm formation compared with drug solution. Incorporation of a positively charged agent into fluid niosomes enhanced the antibacterial activity and reduced biofilm formation significantly. SEM showed evidence of vesicle adsorption to the bacteria with possible adhesion or fusion with the cell membrane. The in vivo skin model confirmed the in vitro results with optimum niosomes being more efficient than drug solution. CONCLUSION: Niosomes are promising for enhanced antibacterial activity and reduced resistance to antibiotics. The later can be achieved by inhibition of biofilm formation.

Egy-score predicts severe hepatic fibrosis and cirrhosis in Egyptians with chronic liver diseases: a pilot study.

BACKGROUND: Non-invasive methods for assessment of hepatic fibrosis are increasingly needed. Recent studies showed that combined elevation of tumor markers CA 19-9 and CA 125 is predictive of severe hepatic fibrosis or cirrhosis with high specificity. OBJECTIVES: We aimed at developing a new panel of surrogate biomarkers for prediction of the stage of hepatic fibrosis by combining tumor markers with other known biomarkers of hepatic fibrosis. PATIENTS AND METHODS: A total of 92 patients with different types of chronic liver diseases (chronic hepatitis B, chronic hepatitis C and autoimmune hepatitis), were prospectively enrolled in our cohort. They were subjected to: ALT, AST, GGT, ALP, total bilirubin, INR, total cholesterol, albumin, platelet count, cancer antigen 19-9 (CA 19-9), cancer antigen 125 (CA 125), cancer antigen 15-3 (CA 15-3), haptoglobin, alpha-2-macroglobulin, apolipoprotein A1, abdominal ultrasound, liver biopsy and histological staging of hepatic fibrosis using the METAVIR system. RESULTS: Combined elevation of CA 19-9 and CA 125 with a summated value > 37 U/mL is predictive of severe hepatic fibrosis or cirrhosis (stage F3-F4 METAVIR) with a probability of 77.6%. Multivariate analysis showed that the most relevant collection of biomarkers for prediction of stage of hepatic fibrosis is: CA 19-9, age, alpha-2- macroglobulin, total bilirubin, platelet count & albumin. We developed a new score, named the "Egy-Score", using a regression equation composed of this panel of biomarkers. Egy-Score could differentiate no or early fibrosis (stage F0-F2 METAVIR) from severe fibrosis or cirrhosis (stage F3-F4 METAVIR) with 83.7% accuracy. CONCLUSIONS: Non-invasive assessment of hepatic fibrosis could be done using the Egy-Score. Egy-Score could differentiate no or early fibrosis (stage F0-F2 METAVIR) from severe fibrosis or cirrhosis (stage F3 - F4 METAVIR) with 83.7% accuracy.

Validated microbiological and HPLC methods for the determination of moxifloxacin in pharmaceutical preparations and human plasma

The article presents a comparison between microbiological and high performance liquid chromatographic (HPLC) assays for quantification of moxifloxacin in tablets, ophthalmic solutions and human plasma. The microbiological method employed a cylinder-plate agar diffusion assay using a strain of Esherichia coli ATCC 25922 as the test organism and phosphate buffer (pH8) as the diluent. The calibration curves were linear (R²> 0.98) over a concentration range of 0.125 to 16 µgml-1. The within day and between days precisions were < 4.47% and < 6.39% respectively. Recovery values were between 89.4 and 110.2%. The HPLC assay used Hypersil® BDS C18 reversed phase column (250×4.6 mm, 5µm) with a mobile phase comprising 20 mM ammonium dihydrogen orthophosphate (pH3) and acetonitrile (75:25) and flowing at 1.5 ml/min. The detection was at 295nm. The calibration curves were linear (R²> 0.999) over the range of 0.125 to 16 µg ml-1. The within day and between days precisions were < 4.07% and < 5.09% respectively. Recovery values were between 97.7 and 107.6%. Similar potencies were obtained after the analysis of moxifloxacin tablets and ophthalmic solutions by both methods. Also pharmacokinetic parameters were calculated after the analysis of plasma samples of six male healthhy volunteers by both validated methods.

validated microbiological and HPLC methods for the determination of moxifloxacin in pharmaceutical preparations and human plasma.

The article presents a comparison between microbiological and high performance liquid chromatographic (HPLC) assays for quantification of moxifloxacin in tablets, ophthalmic solutions and human plasma. The microbiological method employed a cylinder-plate agar diffusion assay using a strain of Esherichia coli ATCC 25922 as the test organism and phosphate buffer (pH8) as the diluent. The calibration curves were linear (R(2) > 0.98) over a concentration range of 0.125 to 16 µgml(-1). The within day and between days precisions were ≤ 4.47% and ≤ 6.39% respectively. Recovery values were between 89.4 and 110.2%. The HPLC assay used Hypersil(®) BDS C18 reversed phase column (250×4.6 mm, 5µm) with a mobile phase comprising 20 mM ammonium dihydrogen orthophosphate (pH3) and acetonitrile (75:25) and flowing at 1.5 ml/min. The detection was at 295nm. The calibration curves were linear (R(2) > 0.999) over the range of 0.125 to 16 µg ml(-1). The within day and between days precisions were ≤ 4.07% and ≤ 5.09% respectively. Recovery values were between 97.7 and 107.6%. Similar potencies were obtained after the analysis of moxifloxacin tablets and ophthalmic solutions by both methods. Also pharmacokinetic parameters were calculated after the analysis of plasma samples of six male healthhy volunteers by both validated methods.