Inhibition of Adherence and Biofilm Formation of Pseudomonas aeruginosa by Immobilized ZnO Nanoparticles on Silicone Urinary Catheter Grafted by Gamma Irradiation.

Nosocomial infections caused by microbial biofilm formation on biomaterial surfaces such as urinary catheters are complicated by antibiotic resistance, representing a common problem in hospitalized patients. Therefore, we aimed to modify silicone catheters to resist microbial adherence and biofilm formation by the tested microorganisms. This study used a simple direct method to graft poly-acrylic acid onto silicone rubber films using gamma irradiation to endow the silicone surface with hydrophilic carboxylic acid functional groups. This modification allowed the silicone to immobilize ZnO nanoparticles (ZnO NPs) as an anti-biofilm. The modified silicone films were characterized by FT-IR, SEM, and TGA. The anti-adherence ability of the modified silicone films was evidenced by the inhibition of biofilm formation by otherwise strong biofilm-producing Gram-positive, Gram-negative, and yeast clinical isolates. The modified ZnO NPs grafted silicone showed good cytocompatibility with the human epithelial cell line. Moreover, studying the molecular basis of the inhibitory effect of the modified silicone surface on biofilm-associated genes in a selected Pseudomonas aeruginosa isolate showed that anti-adherence activity might be due to the significant downregulation of the expression of lasR, lasI, and lecB genes by 2, 2, and 3.3-fold, respectively. In conclusion, the modified silicone catheters were low-cost, offering broad-spectrum anti-biofilm activity with possible future applications in hospital settings.

New insights on Pseudomonas aeruginosa exotoxin A-based immunotoxins in targeted cancer therapeutic delivery.

Pseudomonas aeruginosa exotoxin A-based immunotoxins (PE-ITs) are fusion proteins that harness targeting and toxin moieties. Structural optimizations in PE and targeting moieties were implemented to lower their immunogenicity and alleviate undesirable side effects. PE moiety was engineered to lack its cell-binding domain and T cell epitope regions, whereas single chain (scFv) and disulfide Fv portions (dsFv), nanobodies, and monobodies were utilized as targeting moieties. This review discusses applications of PE-ITs on different types of cancer, structural optimizations to reduce PE-ITs drawbacks, and recent modifications applied for efficient therapeutic delivery. Finally, we draw attention to the possibility of combining radiotherapy, radionuclides, and RGDs with PE-IT to improve overall response rates of IT-based treatments and reduce cancer cell resistance.

Exotoxin A-immunotoxins are proteins that have been used in cancer treatments. The building components of these proteins are very poisonous to both cancer and normal cells. Also, unfavorable body reactions and side effects were seen with their usage. To allow the safe use of these proteins, changes were made in their building components. These changes made them damaging only to cancer cells while being safe to normal non-cancerous cells. This review will talk about the use of exotoxin A-Immunotoxins in different cancer treatments, and how they are created to limit the poisonous effect of their building components to only cancer cells.

Gamma radiation coupled ADP-ribosyl transferase activity of Pseudomonas aeruginosa PE24 moiety.

The ADP-ribosyl transferase activity of P. aeruginosa PE24 moiety expressed by E. coli BL21 (DE3) was assessed on nitrobenzylidene aminoguanidine (NBAG) and in vitro cultured cancer cell lines. Gene encoding PE24 was isolated from P. aeruginosa isolates, cloned into pET22b( +) plasmid, and expressed in E. coli BL21 (DE3) under IPTG induction. Genetic recombination was confirmed by colony PCR, the appearance of insert post digestion of engineered construct, and protein electrophoresis using sodium dodecyl-sulfate polyacrylamide gel (SDS-PAGE). The chemical compound NBAG has been used to confirm PE24 extract ADP-ribosyl transferase action through UV spectroscopy, FTIR, c13-NMR, and HPLC before and after low-dose gamma irradiation (5, 10, 15, 24 Gy). The cytotoxicity of PE24 extract alone and in combination with paclitaxel and low-dose gamma radiation (both 5 Gy and one shot 24 Gy) was assessed on adherent cell lines HEPG2, MCF-7, A375, OEC, and Kasumi-1 cell suspension. Expressed PE24 moiety ADP-ribosylated NBAG as revealed by structural changes depicted by FTIR and NMR, and the surge of new peaks at different retention times from NBAG in HPLC chromatograms. Irradiating recombinant PE24 moiety was associated with a reduction in ADP-ribosylating activity. The PE24 extract IC50 values were < 10 µg/ml with an acceptable R2 value on cancer cell lines and acceptable cell viability at 10 µg/ml on normal OEC. Overall, the synergistic effects were observed upon combining PE24 extract with low-dose paclitaxel demonstrated by the reduction in IC50 whereas antagonistic effects and a rise in IC50 values were recorded after irradiation by low-dose gamma rays. KEY POINTS: • Recombinant PE24 moiety was successfully expressed and biochemically analyzed. • Low-dose gamma radiation and metal ions decreased the recombinant PE24 cytotoxic activity. • Synergism was observed upon combining recombinant PE24 with low-dose paclitaxel.

Bacterial cyclomodulins: types and roles in carcinogenesis.

Various studies confirmed that bacterial infections contribute to carcinogenesis through the excessive accumulation of reactive oxygen species (ROS) and the expression of toxins that disrupt the cell cycle phases, cellular regulatory mechanisms and stimulate the production of tumorigenic inflammatory mediators. These toxins mimic carcinogens which act upon key cellular targets and result in mutations and genotoxicities. The cyclomodulins are bacterial toxins that incur cell cycle modulating effects rendering the expressing bacterial species of high carcinogenic potentiality. They are either cellular proliferating or cell cycle arrest cyclomodulins. Notably, cyclomodulins expressing bacterial species have been linked to different human carcinomas. For instance, Escherichia coli species producing the colibactin were highly prevalent among colorectal carcinoma patients, CagA+ Helicobacter pylori species were associated with MALT lymphomas and gastric carcinomas and Salmonella species producing CdtB were linked to hepatobiliary carcinomas. These species stimulated the overgrowth of pre-existing carcinomas and induced hyperplasia in in vivo animal models suggesting a role for the cyclomodulins in carcinogenesis. Wherefore, the prevalence and mode of action of these toxins were the focus of many researchers and studies. This review discusses different types of bacterial cyclomodulins highlighting their mode of action and possible role in carcinogenesis.

ADP-ribosyl transferase activity and gamma radiation cytotoxicity of Pseudomonas aeruginosa exotoxin A.

This work explores the ADP-ribosyltransferase activity of Pseudomonas (P.) aeruginosa exotoxin A using the guanyl hydrazone derivative, nitrobenzylidine aminoguanidine (NBAG) and the impact of gamma radiation on its efficacy. Unlike the conventional detection methods, NBAG was used as the acceptor of ADP ribose moiety instead of wheat germ extract elongation factor 2. Exotoxin A was extracted from P. aeruginosa clinical isolates and screened for toxA gene using standard PCR. NBAG was synthesized using aminoguanidine bicarbonate and 4-nitrobenzaldehyde and its identity has been confirmed by UV, FTIR, Mass and 13C-NMR spectroscopy. The ADP-ribosyl transferase activity of exotoxin A on NBAG in the presence of Nicotinamide adenine dinucleotide (NAD+) was recorded using UV spectroscopy and HPLC. In vitro ADP-ribosyl transferase activity of exotoxin A protein extract was also explored by monitoring its cytotoxicity on Hep-2 cells using sulforhodamine B cytotoxicity assay. Bacterial broths were irradiated at 5, 10, 15, 24 Gy and exotoxin A protein extract activity were assessed post exposure. Exotoxin A extract exerted an ADP-ribosyltransferase ability which was depicted by the appearance of a new ʎmax after the addition of exotoxin A to NBAG/NAD+ mixture, fragmentation of NAD+ and development of new peaks in HPLC chromatograms. Intracellular enzyme activity was confirmed by the prominent cytotoxic effects of exotoxin A extract on cultured cells. In conclusion, the activity of Exotoxin A can be monitored via its ADP-ribosyltransferase activity and low doses of gamma radiation reduced its activity. Therefore, coupling radiotherapy with exotoxin A in cancer therapy should be carefully monitored.

Effect of sub-inhibitory concentrations of cefepime on biofilm formation by Pseudomonas aeruginosa.

This study investigated the effect of cefepime at sub-minimum inhibitory concentrations (sub-MICs) on in vitro biofilm formation (BF) by clinical isolates of Pseudomonas aeruginosa. The effect of cefepime at sub-MIC levels (½-1/256 MIC) on in vitro BF by six clinical isolates of P. aeruginosa was phenotypically assessed following 24 and 48 h of challenge using the tissue culture plate (TCP) assay. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to observe the change in expression of three biofilm-related genes, namely, a protease-encoding gene (lasA), fimbrial protein-encoding gene (cupA1), and alginate-encoding gene (algC), in a weak biofilm-producing strain of P. aeruginosa following 24 and 48 h of challenge with sub-MICs of cefepime. The BF morphology in response to cefepime was imaged using scanning electron microscopy (SEM). The TCP assay showed strain-, time-, and concentration-dependent changes in in vitro BF in P. aeruginosa following challenge with sub-MICs of cefepime, with a profound increase in strains with inherently no or weak biofilm-producing ability. RT-PCR revealed time-dependent upregulation in the expression of the investigated genes following challenge with ½ and » MIC levels, as confirmed by SEM. Cefepime at sub-MICs could upregulate the expression of BF-related genes and enhance BF by P. aeruginosa clinical isolates.

"Effect of Subinhibitory Concentrations of Some Antibiotics and Low Doses of Gamma Radiation on the Cytotoxicity and Expression of Colibactin by an Uropathogenic Escherichia coli isolate".

Colibactin and cytotoxic necrotizing factor 1 (Cnf 1) are cyclomodulins secreted by uropathogenic E. coli. In this study, uropathogenic E. coli expressing colibactin and Cnf 1 was exposed to antibiotics subMICs and gamma radiation to investigate their effects on its cytotoxicity and expression of colibactin. The test isolate was exposed to three subMIC levels of levofloxacin, ciprofloxacin, trimethoprim/sulfamethoxazole and ceftriaxone and irradiated with gamma rays at 10 and 24.4 Gy. The cytotoxicity for either antibiotic or gamma rays treated cultures was measured using MTT assay and the expression of colibactin encoding genes was determined by RT-PCR. Treatment with fluoroquinolones nearly abolished the cytotoxicity of E. coli isolate and significantly downregulated clbA gene expression at the tested subMICs (P ≤ 0.05) while trimethoprim/sulfamethoxazole treated cultures exerted significant downregulation of clbA and clbQ genes at 0.5 MIC only (P ≤ 0.05). Ceftriaxone treated cultured exhibited reduction in the cytotoxicity and insignificant effects on expression of clbA, clbQ and clbM genes. On contrast, significant upregulation in the expression of clbA and clbQ genes was observed in irradiated cultures (P ≤ 0.05). Fluoroquinolones reduced both the cytotoxicity of UPEC isolate and colibactin expression at different subMICs while ceftriaxone at subMICs failed to suppress the expression of genotoxin, colibactin, giving an insight to the risks associated upon their choice for UTI treatment. Colibactin expression was enhanced by gamma irradiation at doses resembling these received during pelvic radiotherapy which might contribute to post-radiotherapy complications.

Natural outer membrane permeabilizers boost antibiotic action against irradiated resistant bacteria.

BACKGROUND: This study sought to develop new strategies for reverting the resistance of pathogenic Gram-negative bacilli by a combination of conventional antibiotics, potent permeabilizers and natural beta lactamase inhibitors enhancing the activity of various antibiotics. METHODS: The antibiotic susceptibility in the presence of natural non-antibacterial tested concentrations of phytochemicals (permeabilizers and natural beta lactamase inhibitors) was performed by disk diffusion and susceptibility assays. Thymol and gallic acid were the most potent permeabilizers and facilitated the passage of the antibiotics through the outer membrane, as evidenced by their ability to cause LPS release, sensitize bacteria to SDS and Triton X-100. RESULTS: The combination of permeabilizers and natural beta lactamase inhibitors (quercetin and epigallocatechin gallate) with antibiotics induced greater susceptibility of resistant isolates compared to antibiotic treatment with beta lactamase inhibitors alone. Pronounced effects were detected with 24.4 Gy in vitro gamma irradiation on permeability barrier, beta lactamase activity, and outer membrane protein profiles of the tested isolates. CONCLUSIONS: The synergistic effects of the studied natural phytochemicals and antibiotics leads to new clinical choices via outer membrane destabilization (permeabilizers) and/or inactivation of the beta lactamase enzyme, which enables the use of older, more cost-effective antibiotics against resistant strains.

Potential efficacy of garlic lock therapy in combating biofilm and catheter-associated infections; experimental studies on an animal model with focus on toxicological aspects.

BACKGROUND: Life-threatening central venous catheter-related infections are primarily initiated by biofilm formation on the catheter surface. Antibiotic lock therapy is recommended for eradicating intraluminal biofilm. In the era of antibiotic resistance, antibiotics of natural origins provide an effective and cheap option for combating resistant strains. Garlic especially stole the spotlight because of its impressive antimicrobial effectiveness against such superbugs. AIM: Is to estimate the potential use of fresh garlic extract (FGE) as a lock agent against multi-drug resistant (MDR) bacteria. METHODS: The agar well diffusion and broth microdilution techniques were employed to test the antimicrobial activities of FGE against five MDR strains; E. coli, Pseudomonas aeruginosa (P. aeruginosa), Klebsiella pneumoniae (K. pneumoniae), Serratia marscens (S. marscens) and Methicillin-resistant Staphylococcus aureus (MRSA). Then the protective and therapeutic efficiencies of FGE against bacterial biofilms were in-vitro evaluated; at concentrations of 100, 75, 50 and 25%; in tissue culture plate (TCP) and on the polyurethane (PU) sheets using the crystal violet (CV) assay and colony-forming unit (CFU), respectively. Scanning electron microscopy (SEM) was also used to confirm eradication of biofilms on PU sheets. Finally, systemic and deep tissue infections by P. aeruginosa and MRSA were induced in mice that were then treated by FGE at either 100 or 200 mg/kg for seven days. Where the antibacterial activity was assessed by tissue and blood culturing at the end of the treatment period. Biochemical, hematological and histological parameters were also investigated. RESULTS: FGE exhibited potent in-vitro and in-vivo antibacterial and antibiofilm activities against MDR strains. It not only didn't exhibit toxicological effects at the hematological and the histological levels but also provided protective effects as demonstrated by the significant drop in the biochemical parameters. CONCLUSION: FGE has the potential to be used as a prophylactic and/or therapeutic lock agent against biofilm-associated infections caused by MDR bacteria.

Prevalence and pathologic effects of colibactin and cytotoxic necrotizing factor-1 (Cnf 1) in Escherichia coli: experimental and bioinformatics analyses.

BACKGROUND: The colibactin and cytotoxic necrotizing factor 1 (Cnf 1) are toxins with cell cycle modulating effects that contribute to tumorgenesis and hyperproliferation. This study aimed to investigate the prevalence and pathologic effects of Cnf 1 and colibactin among hemolytic uropathogenic Escherichia coli (UPEC). The bioinformatics approach incorporated in this study aimed to expand the domain of the in vitro study and explore the prevalence of both toxins among other bacterial species. A total of 125 E. coli isolates were recovered from UTIs patients. The isolates were tested for their hemolytic activity, subjected to tissue culture and PCR assays to detect the phenotypic and genotypic features of both toxins. A rat ascending UTI in vivo model was conducted using isolates expressing or non-expressing Cnf 1 and colibactin (ClbA and ClbQ). The bioinformatics analyses were inferred by Maximum likelihood method and the evolutionary relatedness was deduced by MEGA X. RESULTS: Only 21 (16.8%) out of 125 isolates were hemolytic and 10 of these (47.62%) harbored the toxins encoding genes (cnf 1 +, clbA + and clbQ +). The phenotypic features of both toxins were exhibited by only 7 of the (cnf 1 + clbA + clbQ +) harboring isolates. The severest infections, hyperplastic and genotoxic changes in kidneys and bladders were observed in rats infected with the cnf 1 + clbA + clbQ + isolates. CONCLUSION: Only 33.3% of the hemolytic UPEC isolates exhibited the phenotypic and genotypic features of Cnf 1 and Colibactin. The in vivo animal model results gives an evidence of active Cnf 1 and Colibactin expression and indicates the risks associated with recurrent and chronic UTIs caused by UPEC. The bioinformatics analyses confirmed the predominance of colibactin pks island among Enterobacteriaceae family (92.86%), with the highest occurrence among Escherichia species (53.57%), followed by Klebsiella (28.57%), Citrobacter (7.14%), and Enterobacter species (3.57%). The Cnf 1 is predominant among Escherichia coli (94.05%) and sporadically found among Shigella species (1.08%), Salmonella enterica (0.54%), Yersinia pseudotuberculosis (1.08%), Photobacterium (1.08%), Moritella viscosa (0.54%), and Carnobacterium maltaromaticum (0.54%). A close relatedness was observed between the 54-kb pks island of Escherichia coli, the probiotic Escherichia coli Nissle 1917, Klebsiella aerogenes, Klebsiella pneumoniae and Citrobacter koseri.